Targeting adenovirus with use of constrained peptide motifs

ABSTRACT

The present invention provides a chimeric adenovirus fiber protein, which differs from the wild-type coat protein by the introduction of a nonnative amino acid sequence in a conformationally-restrained manner. Such a chimeric adenovirus fiber protein according to the invention is able to direct entry into cells of a vector comprising the chimeric fiber protein that is more efficient than entry into cells of a vector that is identical except for comprising a wild-type adenovirus fiber protein rather than the chimeric adenovirus fiber protein. The nonnative amino acid sequences encodes a peptide motif that comprises an epitope for an antibody, or a ligand for a cell surface receptor, that can be employed in cell targeting. The present invention also pertains to vectors comprising such a chimeric adenovirus fiber protein, and to methods of using such vectors.

RELATED APPLICATIONS

[0001] This is continuation application of U.S. patent applicationSerial No. 09/130,225, filed Aug. 6, 1998, which is a divisionalapplication of U.S. patent spplication Ser. No. 08/701,124, filed Aug.21, 1996, now U.S. Pat. No. 5,846,782, which is a continuation-in-partapplication of U.S. patent application Ser. No. 08/563,368, filed Nov.28, 1995, now U.S. Pat. No. 5,965,541.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention pertains to a chimeric adenovirus fiberprotein comprising a constrained nonnative amino acid sequence.

BACKGROUND OF THE INVENTION

[0003] Despite their prior poor reputation as major pathogenic agentsthat lead to numerous infectious diseases, adenoviruses (andparticularly, replication-deficient adenoviruses) have more recentlyattracted considerable recognition as highly effective viral vectors forgene therapy. Adenoviral vectors offer exciting possibilities in thisnew realm of therapeutics based on their high efficiency of genetransfer, substantial carrying capacity, and ability to infect a widerange of cell types (Crystal, Science, 270, 404-410 (1995); Curiel etal., Human Gene Therapy, 3, 147-154 (1992); International PatentApplication WO 95/21259).

[0004] Due to these desirable properties of adenoviruses, recombinantadenoviral vectors have been used for the cell-targeted transfer of oneor more recombinant genes to diseased cells or tissue in need oftreatment. In terms of the general structure of an adenovirus, under theelectron microscope, an adenovirus particle resembles a space capsulehaving protruding antennae (Xia et al., Structure, 2, 1259-1270 (1994)).The viral capsid comprises at least six different polypeptides,including 240 copies of the trimeric hexon (i.e., polypeptide II) and 12copies each of the pentameric penton (polypeptide III) base and trimericfiber (Xia et al., supra).

[0005] An adenovirus uses two separate cellular receptors, both of whichmust be present, to attach to and infect a cell (Wickham et al., Cell,73, 309-319 (1993)). First, the adenovirus fiber protein attaches thevirus to a cell by binding to an as yet unidentified receptor. Then, thepenton base binds to α_(v) integrins, which are a family ofheterodimeric cell-surface receptors that mediate cellular adhesion tothe extracellular matrix molecules, as well as other molecules (Hynes,Cell, 69, 11-25 (1992)). Once an adenovirus is attached to a cell, itundergoes receptor-mediated internalization into clathrin-coatedendocytic vesicles and is stepwise stripped down to the viraldouble-stranded genome, and then the genome (and some accompanying viralcomponents) subsequently is transported to the cell nucleus, thusinitiating infection (Svennson et al., J. Virol., 51, 687-694 (1984);Chardonnet et al., Virology, 40, 462-477 (1970); Greber et al., Cell,75, 477-486 (1993); Fitzgerald et al., Cell, 32, 607-617 (1983)).

[0006] The fiber monomer consists of an amino terminal tail (whichattaches noncovalently to the penton base), a shaft (whose length variesamong different virus serotypes), and a carboxy terminal globular knobdomain (which is necessary and sufficient for host cell binding) (Devauxet al., J. Molec. Biol., 215, 567-588 (1990); xia et al., supra; Greenet al., EMBO J., 2, 1357-1365 (1983); Henry et al., J. Virology, 68(8),5239-5246 (1994)). The regions necessary for trimerization of fiber(which is required for penton base binding) also are located in the knobregion of the protein (Henry et al. (1994), supra; Novelli et al.,Virology, 185, 365-376 (1991)). The fiber, together with the hexon,determine the serotype specificity of the virus, and also comprise themain antigenic determinants of the virus (Watson et al., J. Gen. Virol.,69, 525-535 (1988)).

[0007] This ability of adenoviral fiber and hexon protein to act astargets for a host immune response initially hampered attempts atadenoviral-mediated gene therapy. Namely, alterations in gene expressionmediated by adenovirus are not permanent since the vector is not stablymaintained. However, following adenoviral vector re-administration toprolong the therapeutic response, neutralizing antibodies can be raisedagainst the adenoviral fiber and/or hexon proteins, thus circumventingprotein production (Wohlfart, J. Virology, 62, 2321-2328 (1988);Wohlfart et al., J. Virology, 56, 896-903 (1985)). Fortunately, such animmune response will not be generated with all uses of adenoviralvectors. Similarly, it is now known that if the presence of suchneutralizing antibodies impedes adenoviral-mediated intracellulardelivery, another adenoviral vector, e.g., another serotype adenoviralvector, or another adenovirus vector lacking the epitope against whichthe antibody is directed, can be employed instead (Crompton et al., J.Gen. Virol., 75, 133-139 (1994)). Moreover, newer and effectivetechniques are constantly emerging to prevent an antibody responseagainst the virus from precluding effective re-administration of anadenoviral vector (see, e.g., International Patent Application WO96/12406; Mastrangeli et al., Human Gene Therapy, 7, 79-87 (1996)).

[0008] Thus, adenoviral-mediated gene therapy continues to hold greatpromise, in particular, with respect to redirecting adenovirus tropism.Namely, even though adenovirus can enter an impressive variety of celltypes (see, e.g., Rosenfeld et al., Cell, 68, 143-155 (1992); Quantin etal., Proc. Natl. Acad. Sci., 89, 2581-2584 (1992)); Lemarchand et al,Proc. Natl. Acad. Sci., 89, 6482-6486 (1992); Anton et al., J. Virol.,69, 4600-4606 (1995); LaSalle et al., Science, 259, 988-990 (1993)),there still appear to be cells (e.g., lymphocytes) which are not readilyamenable to adenovirus-mediated gene delivery (see, e.g., Grubb et al.,Nature, 371, 802-806 (1994); Dupuit et al., Human Gene Therapy, 6,1185-1193 (1995); Silver et al., Virology 165, 377-387 (1988); Horvathet al., J. Virol., 62(1), 341-345 (1988)). Similarly, even whentargeting to cells that readily are infected by adenovirus, in manycases, very high levels of adenovirus particles have been used toachieve transduction. This is disadvantageous inasmuch as any immuneresponse associated with adenoviral infection necessarily would beexacerbated with such high levels.

[0009] Accordingly, researchers are seeking new ways to selectivelyintroduce adenoviruses into cells that cannot be infected byadenoviruses, and to increase the effectiveness of adenoviral deliveryinto cells that are infected by adenoviruses. The general principle ofredirecting adenovirus tropism is straightforward. In one commonapproach, by incorporating peptide binding motifs into an adenoviruscoat protein such as fiber protein, the virus can be redirected to binda cell surface binding site that it normally does not bind (see, e.g.,Michael et al., Gene Therapy, 2, 660-668 (1995); International PatentApplication WO 95/26412; International Patent Application WO 94/10323;International Patent Application WO 95/05201). A peptide binding motifis a short sequence of amino acids such as an epitope for an antibody(e.g., a bispecific antibody), or a ligand for a cell surface bindingsite (e.g., a receptor), that can be employed in cell targeting. Whenthe peptide motif binds, for instance to its corresponding cell surfacebinding site to which adenovirus normally does not bind, or binds withonly low affinity, the adenovirus carrying the peptide motif then canselectively deliver genes to the cell comprising this binding site in aspecific and/or more efficient manner.

[0010] However, simply incorporating a known peptide motif into thefiber protein of an adenovirus may not be enough to allow the virus tobind and effectively transduce a target cell. The effectiveness of thepeptide motif in redirecting virus binding to a new cell surface bindingsite depends on multiple factors, including the availability of thepeptide motif to bind to the cell surface receptor, the affinity of thepeptide motif for the cell surface binding site, and the number oftarget binding sites (e.g., receptors) present on the cell targeted forgene delivery. While the lattermost factor currently cannot bemanipulated, in in vivo applications, the former two would appear topresent areas for improvement of prevailing adenoviral-mediated genetherapy. For instance, earlier researchers have not considered that ifthe peptide motif is buried within the structure of the fiber protein,and/or masked by the surrounding structure of the protein, the peptidemotif will not be able to interact with and bind its target. Similarly,previous researchers have not addressed that it is the affinity of thepeptide motif for the cell surface binding site (e.g., receptor) whichdetermines how efficiently the virus can initiate and maintain a bindingcontact with the target receptor, resulting in cellinfection/transduction.

[0011] Thus, there remains a need for improved methods of celltargeting, and adenoviral vectors by which this can be accomplished. Thepresent invention seeks to overcome at least some of the aforesaidproblems of recombinant adenoviral gene therapy. In particular, it is anobject of the present invention to provide improved vectors and methodsfor cell targeting through provision of a chimeric adenovirus fiberprotein comprising a constrained peptide motif. These and other objectsand advantages of the present invention, as well as additional inventivefeatures, will be apparent from the following detailed description.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention provides a chimeric adenoviral fiberprotein which differs from the wild-type (i.e., native) fiber protein bythe introduction of a nonnative amino acid sequence in aconformationally-restrained (i.e., constrained) manner. The introductionresults in the insertion of, or creation of, a constrained peptide motifthat confers upon the resultant chimeric adenovirus fiber protein anability to direct entry into cells of a vector comprising the chimericfiber protein that is more efficient than entry into cells of a vectorthat is identical except for comprising a wild-type adenovirus fiberprotein, and/or an ability to direct entry into cells that adenoviruscomprising the wild-type fiber protein typically does notinfect/transduce. The present invention also provides vectors thatcomprise the chimeric adenovirus fiber protein, and methods ofconstructing and using such vectors.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 is a diagram that illustrates the method of the inventionof targeting adenovirus by conformationally restraining a nonnativeamino acid sequence in an exposed loop of the fiber knob to comprise apeptide binding motif.

[0014]FIG. 2 is a diagram that illustrates the method of the inventionof targeting adenovirus by incorporating a conformationally restrainednonnative amino acid sequence (i.e., a sequence comprising anonpreexisting loop) into the C-terminus of the fiber protein tocomprise a peptide binding motif.

[0015]FIG. 3 is a diagram that depicts the plasmid p193 (F5*) used toconstruct adenovirus fiber chimeras.

[0016]FIG. 4 is a diagram that depicts the plasmid p193 F5F2K, whichencodes a chimeric fiber protein.

[0017]FIG. 5 is a diagram that depicts the plasmid p193 F5F2K(RKKK2),which encodes a chimeric adenovirus fiber protein comprising the heparinbinding domain (i.e., RKKKRKKK [SEQ ID NO:1]) in the exposed HI loop ofthe Ad2 fiber knob.

[0018]FIG. 6 is a diagram that depicts the plasmid p193 F5F2K(FLAG),which encodes a chimeric adenovirus fiber protein comprising the FLAGepitope (i.e., DYKDDDDK [SEQ ID NO:2]) in the exposed HI loop of the Ad2fiber knob.

[0019]FIG. 7 is a bar graph depicting β-galactosidase expression (% ofcontrol) in 293 cells transduced with either AdZ.F5F2K(RKKK2) (closedbars) or AdZ (open bars) in the absence (control) or presence (fiber) ofsoluble fiber protein.

[0020]FIG. 8 depicts the transfer plasmid p193(F5)RGD, which was used tocreate the adenovirus vector AdZ.RGD. FIG. 9 depicts the transferplasmid p193(F5)pLDV, which was used to create the adenovirus vectorAdZ.pLDV.

[0021]FIG. 10 depicts the transfer plasmid p193(F5) pYIGSR, which wasused to create the adenovirus vector AdZ.pYIGSR.

[0022]FIG. 11 is a graph of days post-infection versus FFU/cell for 293cells infected with AdZ (open circles) or AdZ.RGD (closed squares).

[0023]FIG. 12 is a graph of virus particles added (per 6 cm plate)versus β-galactosidase expression (RLU/0.3 μl/7 minutes) for A549 cellsinfected with AdZ (closed circles) or AdZ.RGD (closed triangles).

[0024]FIG. 13 is a graph of virus particles added (per 6 cm plate)versus β-galactosidase expression (RLU/0.3 μl/7 minutes) for CPAE cellsinfected with AdZ (closed circles) or AdZ.RGD (closed triangles).

[0025]FIG. 14 is a graph of virus particles added (per 6 cm plate)versus β-galactosidase expression (RLU/0.3 μl/7 minutes) for HISM cellsinfected with AdZ (closed circles) or AdZ.RGD (closed triangles).

[0026]FIG. 15 is a bar graph depicting the binding of AdZ.RGD (closedbars) and AdZ (open bars) expressed as % of input of cell-bound vectorin 835 kidney cells in either the absence (control) or presence ofcompeting fiber protein (F5), penton base protein (PB), or both fiberand penton base protein (F5/PB).

[0027]FIG. 16 is a bar graph depicting the binding of AdZ.RGD (closedbars) and AdZ (open bars) expressed as % of input of cell-bound vectorin A10 smooth muscle cells in either the absence (control) or presenceof competing fiber protein (F5), penton base protein (PB), or both fiberand penton base protein (F5/PB).

[0028]FIG. 17 is a bar graph depicting the binding of ADZ.RGD (closedbars) and AdZ (open bars) expressed as % of input of cell-bound vectorin CPAE endothelial cells in either the absence (control) or presence ofcompeting fiber protein (F5), penton base protein (PB), or both fiberand penton base protein (F5/PB).

[0029]FIG. 18 is a bar graph depicting β-galactosidase expression (% ofcontrol) in A549 cells transduced with either AdZ.pYIGSR (closed bars)or AdZ (open bars) in the absence (control) or presence (fiber) ofsoluble fiber protein.

[0030]FIG. 19 is a bar graph depicting β-galactosidase expression (% ofcontrol) in Ramos cells transduced with either AdZ.pLDV (closed bars) orAdZ (open bars) in the absence (control) or presence (fiber) of solublefiber protein, or fiber protein and EDTA (fiber +EDTA).

[0031]FIG. 20 is a bar graph depicting β-galactosidase expression (% ofcontrol) in 293 cells transduced with either ADZ.RGD (closed bars),AdZ.pRGD (stippled bars), or AdZ (open bars) in the absence (control) orpresence (fiber) of soluble fiber protein.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides, among other things, a recombinantadenovirus comprising a chimeric fiber protein. The chimeric fiberprotein comprises a constrained nonnative amino acid sequence, inaddition to, or in place of, a native amino acid sequence. Thisnonnative amino acid sequence allows the chimeric fiber (or a vectorcomprising the chimeric fiber) to more efficiently bind to and entercells.

[0033] Chimeric Adenovirus Fiber Protein

[0034] A “fiber protein” according to the invention preferably comprisesan adenoviral fiber protein. Any one of the serotypes of human ornonhuman adenovirus (as described later in the context of the vectorcomprising a chimeric fiber protein) can be used as the source of thefiber protein or fiber gene. Optimally, however, the adenovirus is anAd2 or Ad5 adenovirus.

[0035] The fiber protein is “chimeric” in that it comprises amino acidresidues that are not typically found in the protein as isolated fromwild-type adenovirus (i.e., comprising the native protein, or wild-typeprotein). The fiber protein thus comprises a “nonnative amino acidsequence”. By “nonnative amino acid sequence” is meant a sequence of anysuitable length, preferably from about 3 to about 200 amino acids,optimally from about 3 to about 30 amino acids. Desirably, the nonnativeamino acid sequence is introduced into the fiber protein at the level ofgene expression (i.e., by introduction of a “nucleic acid sequence thatencodes a nonnative amino acid sequence”). Such a nonnative amino acidsequence either is introduced in place of adenoviral sequences, or inaddition to adenoviral sequences. Regardless of the nature of theintroduction, its integration into an adenoviral fiber protein at thelevel of either DNA-or protein, results in the generation of a peptidemotif (i.e., a peptide binding motif) in the resultant chimeric fiberprotein.

[0036] The peptide motif allows for cell targeting, for instance, bycomprising an epitope for an antibody, or a ligand for a cell surfacebinding site. The peptide motif optionally can comprise other elementsof use in cell targeting (e.g., a single-chain antibody sequence). Thepeptide binding motif may be generated by the insertion, and maycomprise, for instance, native and nonnative sequences, or may beentirely made up of nonnative sequences. The peptide motif that resultsfrom the insertion of the nonnative amino acid sequence into thechimeric fiber protein can be either a high affinity peptide (i.e., onethat binds its cognate binding site when provided at a relatively lowconcentration) or a low affinity peptide (i.e., one that binds itscognate binding site when provided at a relatively high concentration).Preferably, however, the resultant peptide motif is a high affinitymotif, particularly one that has become of high affinity for its cognatebinding site due to its constraint within the adenovirus fiber protein.

[0037] An “antibody” includes, but is not limited to, immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules such as portions containing a paratope (i.e., an antigenbinding site). In particular, an antibody preferably can be a bispecificantibody, i.e., having one paratope directed to an epitope of thechimeric fiber protein, and another paratope directed to an epitope of acell surface binding site.

[0038] A “cell surface binding site” encompasses a receptor (whichpreferably is a protein, carbohydrate, glycoprotein, or proteoglycan) aswell as any oppositely charged molecule (i.e., oppositely charged withrespect to the chimeric coat protein) or other type of molecule withwhich the chimeric coat protein can interact to bind the cell, andthereby promote cell entry. Examples of potential cell surface bindingsites include, but are not limited to: heparin and chondroitin sulfatemoieties found on glycosaminoglycans; sialic acid moieties found onmucins, glycoproteins, and gangliosides; major histocompatibilitycomplex I (MHC I) glycoproteins; common carbohydrate components found inmembrane glycoproteins, including mannose, N-acetyl-galactosamine,N-acetyl-glucosamine, fucose, galactose, and the like. However, achimeric fiber protein according to the invention, and methods of usethereof, is not limited to any particular mechanism of cellularinteraction (i.e., interaction with a particular cell surface bindingsite) and is not to be so construed.

[0039] A cell surface binding site according to the invention preferablyis one that previously was inaccessible to interaction with a wild-typeadenoviral fiber protein, or was accessible only at a very low level, asreflected by the reduced efficiency of entry of a wild-type adenoviralfiber protein-containing vector as compared with a vector comprising achimeric adenovirus fiber protein according to the invention. Theinsertion of the nonnative amino acid sequence in the chimeric fiberprotein thus desirably imparts upon the chimeric fiber protein anability to bind to a binding site present on a cell surface whichwild-type fiber protein does not bind, or binds with very low affinity.This preferably results in a situation wherein the chimeric adenovirusfiber protein is able to direct entry into cells of a vector via theinteraction of the normative amino acid sequence, either directly orindirectly, with a cellular receptor other than the fiber receptor.

[0040] This also preferably results in a situation wherein the chimericadenovirus fiber protein is able to direct entry into cells of a vectorcomprising the chimeric adenovirus fiber that is more efficient thanentry into cells of a vector that is identical except for comprising awild-type adenovirus fiber protein rather than the chimeric adenovirusprotein. Also preferably, the chimeric adenovirus fiber protein may actto increase the specificity of targeting, e.g., by changing thespecificity of the fiber protein.

[0041] “Efficiency of entry” can be quantitated by several means. Inparticular, efficiency of entry can be quantitated by introducing achimeric fiber protein into a vector, preferably a viral vector, andmonitoring cell entry (e.g., by vector-mediated delivery to a cell of agene such as a reporter gene) as a function of multiplicity of infection(MOI). In this case, a reduced MOI required for cell entry of a vectorcomprising a chimeric adenoviral fiber protein as compared with a vectorthat is identical, except for comprising a wild-type adenoviral fiberprotein rather than said chimeric adenovirus fiber protein, indicates“more efficient” entry.

[0042] Similarly, efficiency of entry can be quantitated in terms of theability of vectors containing chimeric or wild-type fiber proteins, orthe soluble chimeric or wild-type fiber proteins themselves, to bind tocells. In this case, increased binding exhibited for the vectorcontaining a chimeric adenoviral fiber protein, or the chimeric fiberprotein itself, as compared with the identical vector containing awild-type fiber protein instead, or the wild-type fiber protein itself,is indicative of an increased efficiency of entry, or “more efficient”entry.

[0043] According to this invention, a nonnative amino acid sequence isconformationally-restrained, or “constrained”. A nonnative amino acidsequence is constrained when it is present in a chimeric fiber proteinand is presented to a cell in such a fashion that the ability of thechimeric fiber protein to bind to the cell and/or mediate cell entry isincreased, e.g., relative to the wild-type protein. Such constraintaccording to the present invention can be achieved by the placement of anonnative amino acid sequence in an exposed loop of the chimeric fiberprotein, or, through the placement of the sequence in another locationand creation of a loop-like structure comprising the nonnative aminoacid sequence at that site.

[0044] Adenoviral-mediated gene delivery to specific tissues (i.e., celltargeting) has been impeded by the fact that, generally, lower affinity,unconstrained peptides often are not as effective in mediatingadenovirus binding to target receptors as are constrained peptides. Forinstance, peptide motifs identified by phage display or identified ingenerally are presented in a constrained environment. Accordingly, thepresent application provides a means of targeting adenovirus wherein, inone embodiment, the peptide motifs are presented in the constrainedenvironment of the loop domains of the knob of the adenovirus fiberprotein.

[0045] This method is advantageous since not all the residues of theexposed fiber knob loops are critical for the assembly or functioning ofthe fiber protein, and thus provide convenient sites at which thepeptide motifs can be inserted. This method further is advantageous inthat additions within a loop of a protein structure will be moreresistant to proteolytic degradation than will additions in the end of aprotein. Moreover, for low affinity peptide motifs in particular, thismethod is more efficient than the method wherein the peptide motifs arepresented as unconstrained linear structures at the C-terminus of theknob of the fiber. Conceivably, “constraint”, according to theinvention, increases affinity since it puts the molecule in atopological conformation in which it is in sync with its receptor, and,in this fashion, facilitates binding. However, the specification is notlimited to any particular mechanism of action and is not to be soconstrued.

[0046] In terms of the loop domains of the fiber knob which can beemployed in the context of the invention, the crystal structure of thefiber knob has been described (see, e.g., Xia et al., supra,particularly FIG. 4). The knob monomer comprises an eight-strandedantiparallel β-sandwich fold. The overall structure of the fiber knobtrimer resembles a three-bladed propeller with certain β-strands of eachof the three monomers comprising the faces of the blades. In particular,the following residues of the Ad5 fiber knob appear important inhydrogen bonding in the β-sandwich motif: 400-402, 419-428, 431-440,454-461, 479-482, 485-486, 516-521, 529-536, 550-557, and 573-578. Theremaining residues of the protein (which do not appear to be critical informing the fiber protein secondary structure) define the exposed loopsof the protein knob domain. In particular, residues inclusive of 403-418comprise the AB loop, residues inclusive of 441-453 comprise the CDloop, residues inclusive of 487-514 comprise the DG loop, residuesinclusive of 522-528 comprise the GH loop, residues inclusive of 537-549comprise the HI loop and residues inclusive of 558-572 comprise the IJloop.

[0047] According to this invention, “loop” is meant in the generic senseof defining a span of amino acid residues (i.e., more than one,preferably less than two hundred, and even more preferably, less thanthirty) that can be substituted by the nonnative amino acid sequence tocomprise a peptide motif that allows for cell targeting. While suchloops are defined herein with respect to the Ad5 sequence, the sequencealignment of other fiber species have been described (see, e.g., Xia etal., supra). For these other species (particularly Ad2, Ad3, Ad7, Ad40and Ad41 described in Xia et al., supra), the corresponding loop regionsof the knob domains appear to be comparable.

[0048] Furthermore, the corresponding residues important in the fiberknob for protein binding/folding appear to be conserved between fiberproteins of different adenoviral serotypes (Xia et al., supra). Thissuggests that even for those adenoviral species in which the crystalstructure of the fiber protein is not known, outside of these conservedresidues will lie nonconserved regions, or regions that do not exhibitthe high level of conservation observed for the residues critical toprotein functionality. Likely the sequence of the fiber knob protein inthese nonconserved regions will be present as a loop due to the absenceof important intramolecular interactions in this region of the protein.The loop sequences comprising these nonconserved regions similarly canbe mutated as described herein by incorporation of peptide motifsallowing cell targeting. These so-called non-conserved sequences likelyinclude any amino acids that occur outside of the conserved regions(i.e., residues noninclusive of those corresponding to AdS residues400-402, 419-428, 431-440, 454461, 479-482, 485-486, 516-521, 529-536,550-557, and 573-578).

[0049] More generally, the nonconserved regions will comprisehydrophobic residues that typically are found on the interior of aprotein. Such hydrophobic residues include, but are not limited to, Ile,Val, Leu, Trp, Cys, and Phe. In contrast, the conserved regionsgenerally will comprise hydrophilic residues such as charged residues(e.g., Arg, Lys, Glu, Asp, and the like) or polar residues or residuescomprising a hydroxyl group (e.g., Thr, Ser, Asn, Gln, etc.). This meansthat a rough approximation of the exposed and buried amino acids of thefiber protein can be derived based on its hydrophobicity/hydrophilicityplot.

[0050] Thus, the present invention preferably provides a chimericadenovirus fiber protein comprising a constrained nonnative amino acidsequence. Preferably, the nonnative amino acid sequence is constrainedby its presence in a loop of the knob of the chimeric fiber protein. Inparticular, desirably the nonnative amino acid sequence is inserted intoor in place of a protein sequence in a loop of the knob of the chimericadenoviral fiber protein. Optionally, the fiber protein loop is selectedfrom the group consisting of the AB, CD, DG, GH, and IJ loops, anddesirably is the HI loop. Also, preferably, the loop comprises aminoacid residues in the fiber knob other than Ad5 residues 400-402, 419428,431-440, 454-461, 479-482, 485-486, 516-521, 529-536, 550-557, and573-578. Desirably, the loop comprises amino acid residues selected fromthe group consisting of residues 403-418, 441-453, 487-514, 522-528,537-549, and 558-572.

[0051] In particular, preferably the nonnative amino acid sequencepresent in the loop compnses a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:31, SEQ IDNO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:53, SEQ IDNO:56, SEQ ID NO:59, and SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:67, SEQID NO:68, SEQ ID NO:86, and wherein the sequence may be deleted ateither the C- or N-terminus by 1, 2, or 3 residues. The nonnative aminoacid sequence also desirably can comprise conservative amino acidsubstitutions of these sequences, as further described herein.Optionally, these sequences can be present in the chimeric protein asdepicted, for instance, in FIG. 4, FIG. 5, FIG. 6, FIG. 8, FIG. 9, andFIG. 10.

[0052] The invention also provides a means of targeting adenoviruswherein the peptide motifs are presented in a constrained environment atthe C-terminus of the fiber protein in the region of the fiber knob.This method entails the generation of loops (i.e., “nonpreexistingloops”) by bonding between cysteine residues or through use of othersequences capable of forming loops (e.g., a β-sheet), thereby creating aloop-like secondary structure in the domain of the protein in which thepeptide motif is inserted. Generally, according to the invention, thenonnative amino acid sequence being added itself will form a loop-likestructure (e.g., through disulfide bonding between cysteine residuesoccurring in vivo). However, it also is possible that the loop may formdue to bonding, e.g., between a cysteine residue present in thenonnative amino acid sequence, and one in the wild-type fiber protein.In this sense, the looping of the sequence is not inherent, but ispotential.

[0053] In particular, a chimeric adenovirus fiber protein according tothe invention comprises a nonnative amino acid sequence that isconstrained, preferably by its possession of an RGD peptide (or othersimilar peptide such as LDV, as described herein) and one or morecysteine pairs. According to this invention, a “pair” comprises twocysteines separated by at least one intervening amino acid. Desirably,when the sequence comprises only a single pair, the cysteines areseparated by the RGD sequence (or other similar sequence that can beemployed to effect cell targeting, and preferably, is less than 30 aminoacids) such that the nonpreexisting loop can be created, i.e., throughdisulfide bonding. Preferably, the cysteine residues in this case areseparated by less than 30 amino acids, for instance, a mixture ofglycine and serine residues as in [SEQ ID NO:73]. Regardless of thenonnative amino acid sequence employed, it must comprise a loop-likesecondary structure.

[0054] In terms of this nonpreexisting loop, one potential peptide motifand variations thereof have been described herein. However, otherRGD-containing cyclic peptides have been described in the literature andcan be employed in the context of the invention as the nonnative aminoacid sequence (see, e.g., Koivunen et al., Bio/Technology, 13, 265-270(1995)). In particular, another nonnative amino acid sequence accordingto the invention can comprise the sequence CDCRGDCFC ([SEQ ID NO:3]. Thenonnative amino acid sequence, however, preferably comprises Cys Xaa CysArg Gly Asp Cys Xaa Cys [SEQ ID NO:4] (wherein “Xaa” is any nucleicacid) or Cys(Xaa)_(A) Cys Arg Gly Asp Cys(Xaa)_(B) Cys, wherein “A” and“B” can vary independently and can be any number from 0 to 8, so long aseither A or B is 1. In particular, the nonnative amino acid sequencepreferably comprises the sequence Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys Arg Gly Asp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys [SEQ ID NO:5],wherein deletions can be made of amino acid residues other than cysteineon either one or both side(s) of the RGD (i.e., Arg Gly Asp) sequence of1, 2, 3, 4, 5, 6, 7, or 8 residues.

[0055] Thus, desirably the nonnative amino acid sequence comprising thenonpreexisting loop is inserted into or in place of a protein sequenceat the C-terminus of the chimeric adenovirus fiber protein. Preferablythe nonnative amino acid sequence comprising the nonpreexisting loop isinserted into a loop of the knob of the chimeric adenoviral fiberprotein. Optimally the nonnative amino acid sequence comprises asequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4and SEQ ID NO:5, wherein the sequence may be deleted at either the C- orN-terminus by 1, 2, or 3 residues. The amino acid sequence alsodesirably can comprise conservative amino acid substitutes of thesesequences, as further described herein.

[0056] The nonpreexisting loop optionally is attached to the C-terminusof the fiber protein or in a fiber knob loop by means of a so-called“spacer” sequence. The spacer sequence may comprise part of thenonnative amino acid sequence proper, or it may be an entirely separatesequence. In particular, a spacer sequence is a sequence that preferablyintervenes between the native protein sequence and the nonnativesequence, between a nonnative sequence and another nonnative sequence,or between a native sequence and another native sequence. Such asequence desirably is incorporated into the protein to ensure that thenonnative sequence comprising the epitope for an antibody or cellsurface binding site projects from the three dimensional structure ofthe chimeric fiber in such a fashion so as to be able to interact withand bind to cells. A spacer sequence can be of any suitable length,preferably from about 3 to about 30 amino acids, and comprises any aminoacids, for instance, a mixture of glycine and serine residues as in [SEQID NO:73]. Optimally, the spacer sequence does not interfere with thefunctioning of the fiber protein.

[0057] Nucleic Acid Encoding a Chimeric Adenovirus Fiber Protein

[0058] As indicated previously, preferably the nonnative amino acidsequence is introduced at the level of DNA. Accordingly, the inventionalso provides an isolated and purified nucleic acid encoding a chimericadenovirus fiber protein comprising a constrained nonnative amino acidsequence according to the invention. Desirably, the nucleic acidsequence that encodes the nonnative amino acid sequence comprises asequence selected from the group consisting of SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:52, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:62, as well asconservatively modified variants of these nucleic acid sequences.

[0059] A “conservatively modified variant” is a variation on the nucleicacid sequence that results in a conservative amino acid substitution. A“conservative amino acid substitution” is an amino acid substituted byan alternative amino acid of similar charge density,hydrophilicity/hydrophobicity, size, and/or configuration (e.g., Val forIle). In comparison, a “nonconservatively modified variant” is avariation on the nucleic acid sequence that results in a nonconservativeamino acid substitution. A “nonconservative amino acid substitution” isan amino acid substituted by an alternative amino acid of differingcharge density, hydrophilicity/hydrophobicity, size, and/orconfiguration (e.g., Val for Phe). The means of making suchmodifications are well known in the art, are described in the Exampleswhich follow, and also can be accomplished by means of commerciallyavailable kits and vectors (e.g., New England Biolabs, Inc., Beverly,Mass.; Clontech, Palo Alto, Calif.). Moreover, the means of assessingsuch substitutions (e.g., in terms of effect on ability to bind andenter cells) are described in the Examples herein. Other approachesdescribed in the art also are available for identifying peptidesequences that can act as ligands for a cell surface receptor and,hence, are of use in the present invention (see, e.g., Russell, NatureMedicine, 2, 276-277 (1996)).

[0060] The means of making such a chimeric fiber protein, particularlythe means of introducing the sequence at the level of DNA, is well knownin the art, and is described in the Examples that follow. Briefly, themethod comprises introducing a sequence into the sequence encoding thefiber protein so as to insert a new peptide motif into or in place of aprotein sequence at the C-terminus of the wild-type fiber protein, or ina loop of a knob of the wild-type fiber protein. Such introduction canresult in the insertion of a new peptide binding motif, or creation of apeptide motif (e.g., wherein some of the sequence comprising the motifis already present in the native fiber protein). The method also can becarried out to replace fiber sequences with a nonnative amino acidsequence according to the invention.

[0061] Generally, this can be accomplished by cloning the nucleic acidsequence encoding the chimeric fiber protein into a plasmid or someother vector for ease of manipulation of the sequence. Then, a uniquerestriction site at which further sequences can be added into the fiberprotein is identified or inserted into the fiber sequence. Adouble-stranded synthetic oligonucleotide generally is created fromoverlapping synthetic single-stranded sense and antisenseoligonucleotides such that the double-stranded oligonucleotideincorporates the restriction sites flanking the target sequence and, forinstance, can be used to incorporate replacement DNA. The plasmid orother vector is cleaved with the restriction enzyme, and theoligonucleotide sequence having compatible cohesive ends is ligated intothe plasmid or other vector to replace the wild-type DNA. Other means ofin vitro site-directed mutagenesis such as are known to those skilled inthe art, and can be accomplished (in particular, using PCR), forinstance, by means of commercially available kits, can also be used tointroduce the mutated sequence into the fiber protein coding sequence.

[0062] Once the mutated sequence is introduced into the chimeric coatprotein, the nucleic acid fragment encoding the sequence can beisolated, e.g., by PCR amplification using 5′ and 3′ primers, preferablyones that terminate in further unique restriction sites. Use of primersin this fashion results in an amplified chimeric fiber-containingfragment that is flanked by the unique restriction sites. The uniquerestriction sites can be used for further convenient subcloning of thefragment. Other means of generating a chimeric fiber protein also can beemployed. These methods are highly familiar to those skilled in the art.

[0063] Vector Comprising a Chimeric Adenovirus Fiber Protein

[0064] A “vector” according to the invention is a vehicle for genetransfer as that term is understood by those skilled in the art. Threetypes of vectors encompassed by the invention are: plasmids, phages, andviruses. Plasmids, phages, and viruses can be transferred to a cell intheir nucleic acid form (e.g., via transfection). In comparison, phagesand viruses also can be transferred with the nucleic acid in a“capsular” form. Hence, the vectors (e.g., capsular form) that can beemployed for gene transfer are referred to herein generally as“vectors”, with nucleic acid forms being referred to more particularlyas “transfer vectors”. However, transfer vectors also are vectors withinthe context of the invention.

[0065] Preferably, a vector according to the invention is a virus,especially a virus selected from the group consisting of nonenvelopedviruses, i.e., nonenveloped RNA or DNA viruses. Also, a virus can beselected from the group consisting of enveloped viruses, i.e., envelopedRNA or DNA viruses. Such viruses preferably comprise a fiber protein, oran analogous coat protein that is used for cell entry. Desirably, theviral coat protein is one that projects outward from the capsid suchthat it is able to interact with cells. In the case of enveloped RNA orDNA viruses, preferably the coat protein is a lipid envelopeglycoprotein (i.e., a so-called spike or peplomer).

[0066] In particular, preferably a vector is a nonenveloped virus (i.e.,either a RNA or DNA virus) from the family Hepadnaviridae, Parvoviridae,Papovaviridae, Adenoviridae, or Picornaviridae. A preferred nonenvelopedvirus according to the invention is a virus of the familyHepadnaviridae, especially of the genus Hepadnavirus. A virus of thefamily Parvoviridae desirably is of the genus Parvovirus (e.g.,parvoviruses of mammals and birds) or Dependovirus (e.g.,adeno-associated viruses (AAVs)). A virus of the family Papovaviridaepreferably is of the subfamily Papillomavirinae (e.g., thepapillomaviruses including, but not limited to, human papillomaviruses(HPV) 1-48) or the subfamily Polyomavirinae (e.g., the polyomavirusesincluding, but not limited to, JC, SV40 and BK virus). A virus of thefamily Adenoviridae desirably is of the genus Mastadenovirus (e.g.,mammalian adenoviruses) or Aviadenovirus (e.g., avian adenoviruses). Avirus of the family Picornaviridae is preferably a hepatitis A virus(HAV), hepatitis B virus (HBV), or a non-A or non-B hepatitis virus.

[0067] Similarly, a vector can be an enveloped virus from the familyHerpesviridae or Retroviridae, or can be a Sindbis virus. A preferredenveloped virus according to the invention is a virus of the familyHerpesviridae, especially of the subfamily or genus Alphaherpesvirinae(e.g., herpes simplex-like viruses), Simplexvirus (e.g., herpessimplex-like viruses), Varicellavirus (e.g., varicella andpseudorabies-like viruses), Betaherpesvirinae (e.g., thecytomegaloviruses), Cytomegalovirus (e.g., the human cytomegaloviruses),Gammaherpesvirinae (e.g., the lymphocyte-associated viruses), andLymphocryptovirus (e.g., EB-like viruses).

[0068] Another preferred enveloped virus is a RNA virus of the familyRetroviridae (i.e., a retrovirus), particularly a virus of the genus orsubfamily Oncovirinae, Spumavirinae, Spumavirus, Lentivirinae, orLentivirus. A RNA virus of the subfamily Oncovirinae is desirably ahuman T-lymphotropic virus type 1 or 2 (i.e., HTLV-1 or HTLV-2) orbovine leukemia virus (BLV), an avian leukosis-sarcoma virus (e.g., Roussarcoma virus (RSV), avian myeloblastosis virus (AMV), avianerythroblastosis virus (AEV), Rous-associated virus (RAV)-1 to 50,RAV-0), a mammalian C-type virus (e.g., Moloney murine leukemia virus(MuLV), Harvey murine sarcoma virus (HaMSV), Abelson murine leukemiavirus (A-MuLV), AKR-MuLV, feline leukemia virus (FeLV), simian sarcomavirus, reticuloendotheliosis virus (REV), spleen necrosis virus (SNV)),a B-type virus (e.g., mouse mammary tumor virus (MMTV)), or a D-typevirus (e.g., Mason-Pfizer monkey virus (MPMV), “SAIDS” viruses). A RNAvirus of the subfamily Lentivirus is desirably a human immunodeficiencyvirus type 1 or 2 (i.e., HIV-1 or HIV-2, wherein HIV-1 was formerlycalled lymphadenopathy associated virus 3 (HTLV-III) and acquired immunedeficiency syndrome (AIDS)-related virus (ARV)), or another virusrelated to HIV-1 or HIV-2 that has been identified and associated withAIDS or AIDS-like disease. The acronym “HIV” or terms “AIDS virus” or“human immunodeficiency virus” are used herein to refer to these HIVviruses, and HIV-related and -associated viruses, generically. Moreover,a RNA virus of the subfamily Lentivirus preferably is a Visna/maedivirus (e.g., such as infect sheep), a feline immunodeficiency virus(FIV), bovine lentivirus, simian immunodeficiency virus (SIV), an equineinfectious anemia virus (EIAV), or a caprine arthritis-encephalitisvirus (CAEV).

[0069] An especially preferred vector according to the invention is anadenoviral vector (i.e., a viral vector of the family Adenoviridae,optimally of the genus Mastadenovirus). Desirably such a vector is anAd2 or Ad5 vector, although other serotype adenoviral vectors can beemployed. Adenoviral stocks that can be employed according to theinvention include any of the adenovirus serotypes 1 through 47 currentlyavailable from American Type Culture Collection (ATCC, Rockville, Md.),or from any other serotype of adenovirus available from any othersource. For instance, an adenovirus can be of subgroup A (e.g.,serotypes 12, 18, 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21,34, 35), subgroup C (e.g., serotypes 1, 2, 5, 6), subgroup D (e.g.,serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-47),subgroup E (serotype 4), subgroup F (serotype 40, 41), or any otheradenoviral serotype.

[0070] The adenoviral vector employed for gene transfer can be wild-type(i.e., replication competent). Alternately, the adenoviral vector cancomprise genetic material with at least one modification therein, whichcan render the virus replication deficient. The modification to theadenoviral genome can include, but is not limited to, addition of a DNAsegment, rearrangement of a DNA segment, deletion of a DNA segment,replacement of a DNA segment, or introduction of a DNA lesion. A DNAsegment can be as small as one nucleotide and as large as 36 kilobasepairs (i.e., the approximate size of the adenoviral genome) or,alternately, can equal the maximum amount which can be packaged into anadenoviral virion (i.e., about 38 kb). Preferred modifications to theadenoviral genome include modifications in the E1, E2, E3 and/or E4region. An adenoviral vector also preferably can be a cointegrated,i.e., a ligation of adenoviral genomic sequences with other sequences,such as other virus, phage, or plasmid sequences.

[0071] In terms of a viral vector (e.g., particularly a replicationdeficient adenoviral vector), such a vector can comprise either completecapsids (i.e., including a viral genome such as an adenoviral genome) orempty capsids (i.e., in which a viral genome is lacking, or is degraded,e.g., by physical or chemical means). Preferably the viral vectorcomprises complete capsides. Along the same lines, since methods areavailable for transferring viruses, plasmids, and phages in the form oftheir nucleic acid sequences (i.e., RNA or DNA), a vector (i.e., atransfer vector) similarly can comprise RNA or DNA, in the absence ofany associated protein such as capsid protein, and in the absence of anyenvelope lipid. Thus, according to the invention whereas a vector“comprises” a chimeric adenoviral fiber protein, a transfer vectorcomprises a chimeric adenoviral fiber protein in the sense that it“encodes” the chimeric adenoviral fiber protein.

[0072] A vector according to the invention can comprise additionalsequences and mutations, e.g., some within the fiber protein itself. Forinstance, a vector according to the invention further preferablycomprises a nucleic acid comprising a passenger gene.

[0073] A “nucleic acid” is a polynucleotide (DNA or RNA). A “gene” isany nucleic acid sequence coding for a protein or a nascent RNAmolecule. A “passenger gene” is any gene which is not typically presentin and is subcloned into a vector (e.g., a transfer vector) according tothe present invention, and which upon introduction into a host cell isaccompanied by a discernible change in the intracellular environment(e.g., by an increased level of deoxyribonucleic acid (DNA), ribonucleicacid (RNA), peptide or protein, or by an altered rate of production ordegradation thereof). A “gene product” is either an as yet untranslatedRNA molecule transcribed from a given gene or coding sequence (e.g.,MRNA or antisense RNA) or the polypeptide chain (i.e., protein orpeptide) translated from the MRNA molecule transcribed from the givengene or coding sequence. Whereas a gene comprises coding sequences plusany non-coding sequences, a “coding sequence” does not include anynon-coding (e.g., regulatory) DNA. A gene or coding sequence is“recombinant” if the sequence of bases along the molecule has beenaltered from the sequence in which the gene or coding sequence istypically found in nature, or if the sequence of bases is not typicallyfound in nature. According to this invention, a gene or coding sequencecan be wholly or partially synthetically made, can comprise genomic orcomplementary DNA (cDNA) sequences, and can be provided in the form ofeither DNA or RNA.

[0074] Non-coding sequences or regulatory sequences include promotersequences. A “promoter” is a DNA sequence that directs the binding ofRNA polymerase and thereby promotes RNA synthesis. “Enhancers” arecis-acting elements of DNA that stimulate or inhibit transcription ofadjacent genes. An enhancer that inhibits transcription is also termed a“silencer”. Enhancers differ from DNA-binding sites forsequence-specific DNA binding proteins found only in the promoter (whichare also termed “promoter elements”) in that enhancers can function ineither orientation, and over distances of up to several kilobase pairs,even from a position downstream of a transcribed region. According tothe invention, a coding sequence is “operably linked” to a promoter(e.g., when both the coding sequence and the promoter constitute apassenger gene) when the promoter is capable of directing transcriptionof that coding sequence.

[0075] Accordingly, a “passenger gene” can be any gene, and desirably iseither a therapeutic gene or a reporter gene. Preferably a passengergene is capable of being expressed in a cell in which the vector hasbeen internalized. For instance, the passenger gene can comprise areporter gene, or a nucleic acid sequence which encodes a protein thatcan in some fashion be detected in a cell. The passenger gene also cancomprise a therapeutic gene, for instance, a therapeutic gene whichexerts its effect at the level of RNA or protein. For instance, aprotein encoded by a transferred therapeutic gene can be employed in thetreatment of an inherited disease, such as, e.g., the cystic fibrosistransmembrane conductance regulator cDNA for the treatment of cysticfibrosis. The protein encoded by the therapeutic gene may exert itstherapeutic effect by resulting in cell killing. For instance,expression of the gene in itself may lead to cell killing, as withexpression of the diphtheria toxin A gene, or the expression of the genemay render cells selectively sensitive to the killing action of certaindrugs, e.g., expression of the HSV thymidine kinase gene renders cellssensitive to antiviral compounds including acyclovir, gancyclovir andFIAU (1-(2-deoxy-2-fluoro-β-D-arabinofuranosil)-5-iodouracil).

[0076] Moreover, the therapeutic gene can exert its effect at the levelof RNA, for instance, by encoding an antisense message or ribozyme, aprotein which affects splicing or 3′ processing (e.g., polyadenylation),or can encode a protein which acts by affecting the level of expressionof another gene within the cell (i.e., where gene expression is broadlyconsidered to include all steps from initiation of transcription throughproduction of a processed protein), perhaps, among other things, bymediating an altered rate of mRNA accumulation, an alteration of mRNAtransport, and/or a change in post-transcriptional regulation.Accordingly, the use of the term “therapeutic gene” is intended toencompass these and any other embodiments of that which is more commonlyreferred to as gene therapy as known to those of skill in the art.Similarly, the recombinant adenovirus can be used for gene therapy or tostudy the effects of expression of the gene in a given cell or tissue invitro or in vivo.

[0077] The present invention accordingly provides a vector comprising achimeric adenovirus fiber protein that comprises a constrained nonnativeamino acid sequence. Such a vector preferably comprises a passenger genewhich optionally is either inserted into the adenoviral genome or isattached to a coat protein (i.e., penton base, fiber, or hexon protein)of the adenovirus by means of a protein/DNA interaction. Alternately,the adenoviral vector preferably carries into a cell an unlinked DNA orprotein molecule, or other small moiety, by means of adenovirusbystander-mediated uptake of these molecules (International PatentApplication WO 95/21259).

[0078] Along these lines, the method of the invention can be employed totransfer nucleic acid sequences which are transported as part of theadenoviral genome (i.e., encoded by adenovirus), and to transfer nucleicacid sequences that are attached to the outside of the adenoviral capsid(Curiel et al., supra), as well as unattached DNA, protein, or othersmall molecules that similarly can be transported by adenoviralbystander-mediated uptake (International Patent Application WO95/21259). The method can be employed to mediate gene and/or proteindelivery either ex vivo or in vivo, as described herein.

[0079] Desirably, a vector is a viral vector selected from the groupconsisting of nonenveloped viruses. Such a vector desirably comprises anonnative amino acid sequence according to the invention and/or anucleic acid sequence that encodes such nonnative amino acid sequence.Optimally, the vector is an adenoviral vector, particularly anadenoviral vector selected from the group consisting of AdZ.FLAG,AdZ.RKKK2, AdZ.pGS, AdZ.RGD, AdZ.pRGD, AdZ.pLDV, and AdZ.pYIGSR.

[0080] The means of making the recombinant adenoviral vectors accordingto the invention are known to those skilled in the art. For instance,recombinant adenovirus comprising a chimeric fiber protein and therecombinant adenovirus that additionally comprises a passenger gene orgenes capable of being expressed in a particular cell can be generatedby use of a transfer vector, preferably a viral or plasmid transfervector, in accordance with the present invention. Such a transfer vectorpreferably comprises a chimeric adenoviral fiber sequence as previouslydescribed. The chimeric fiber protein gene sequence comprises anonnative (i.e., non-wild-type) sequence in place of the nativesequence, which has been deleted, or in addition to the native sequence.

[0081] A recombinant chimeric fiber protein gene sequence can be movedto or from an adenoviral vector from or into a baculovirus or a suitableprokaryotic or eukaryotic expression vector for expression andevaluation of receptor or protein specificity and avidity, trimerizationpotential, penton base binding, and other biochemical characteristics.In particular, the method of protein production in baculovirus as setforth in the Examples which follow, and as described in Wickham et al.(1995), supra, can be employed.

[0082] Accordingly, the present invention also provides recombinantbaculoviral and prokaryotic and eukaryotic expression vectors comprisinga chimeric adenoviral fiber protein gene sequence, which also can betransfer vectors. The present invention also provides vectors that fallunder a commonly employed definition of transfer vectors, e.g., vectorswhich are plasmids containing adenovirus sequences that are used tocreate new adenovirus vectors. The chimeric fiber protein gene sequenceincludes a nonnative sequence in addition to or in place of a nativeamino acid sequence. This enables the resultant chimeric fiber proteinto bind to a binding site other than a binding site bound by the nativesequence. By moving the chimeric gene from an adenoviral transfer vectorto baculovirus or a prokaryotic or eukaryotic expression vector, highprotein expression is achievable (approximately 5-50% of the totalprotein being the chimeric fiber). Preferred transfer vectors accordingto the invention are selected from the group consisting of p193(F5*),p193 F5F2K(FLAG), p193 F5F2K, p193 F5F2K(RKKK2), p193(F5)pGS(RGD),p193(F5)pLDV, p193(F5)pYIGSR, and p193(F5*)RGD.

[0083] A vector according to the invention further can comprise, eitherwithin, in place of, or outside of the coding sequence of a fiberprotein additional sequences that impact upon the ability of the fiberprotein to trimerize, or comprise a protease recognition sequence. Asequence that impacts upon the ability to trimerize is one or moresequences that enable fiber trimerization. A sequence that comprises aprotease recognition sequence is a sequence that can be cleaved by aprotease, thereby effecting removal of the chimeric coat protein (or aportion thereof) and attachment of the recombinant adenovirus to a cellby means of another coat protein. When employed with a fiber protein,the protease recognition site preferably does not affect fibertrimerization or receptor specificity of the fiber protein. Forinstance, in one embodiment of the present invention, preferably thefiber protein, or a portion thereof, is deleted by means of a proteaserecognition sequence, and then the penton base protein, or anotherprotein, commands cell binding/cell entry.

[0084] In terms of the production of vectors and transfer vectorsaccording to the invention, transfer vectors are constructed usingstandard molecular and genetic techniques such as are known to thoseskilled in the art. Vectors comprising virions or virus particles areproduced using viral vectors in the appropriate cell lines. Similarly,the adenoviral fiber chimera-containing particles are produced instandard cell lines, e.g., those currently used for adenoviral vectors.Following production and purification, the particles in which fiber isto be deleted are rendered fiberless through digestion of the particleswith an appropriate sequence-specific protease, which cleaves the fiberproteins and releases them from the viral particles to generatefiberless particles.

[0085] Illustrative Uses

[0086] The present invention provides a chimeric fiber protein that isable to bind to cells and mediate entry into cells with high efficiency,as well as vectors and transfer vectors comprising same. The chimericfiber protein itself has multiple uses, e.g., as a tool for studies invitro of adenovirus binding to cells (e.g., by Scatchard analysis asshown previously by Wickham et al. (1993), supra), to block binding ofadenovirus to receptors in vitro (e.g., by using antibodies, peptides,and enzymes, as described in the Examples herein and as known in theart), and, with use of some chimeric fiber proteins comprisingparticular peptide motifs, to protect against adenoviral infection invivo by competing for binding to the binding site by which adenoviruseffects cell entry.

[0087] A vector comprising a chimeric fiber protein also can be used instrain generation and as a means of making new vectors. For instance,the nonnative amino acid sequence can be introduced intracellularly as ameans of generating new vectors via recombination. Similarly, a vectorcan be used in gene therapy. For instance, a vector of the presentinvention can be used to treat any one of a number of diseases bydelivering to targeted cells corrective DNA, i.e., DNA encoding afunction that is either absent or impaired, or a discrete killing agent,e.g., DNA encoding a cytotoxin that, for example, is active onlyintracellularly. Diseases that are candidates for such treatmentinclude, for example, cancer, e.g., melanoma, glioma or lung cancers;genetic-disorders, e.g., cystic fibrosis, hemophilia or musculardystrophy; pathogenic infections, e.g., human immunodeficiency virus,tuberculosis or hepatitis; heart disease, e.g., preventing restenosisfollowing angioplasty or promoting angiogenesis to reperfuse necrotictissue; and autoimmune disorders, e.g., Crohn's disease, colitis orrheumatoid arthritis.

[0088] In particular, gene therapy can be carried out in the treatmentof diseases, disorders, or conditions associated with different tissuesthat, prior to the present invention, adenovirus was not able to bind toand enter, or could do so only with low affinity and/or specificity. Forinstance, the method can be employed to incorporate a targeting sequencewhich permits an increased efficiency of gene delivery to differenttissues. Such targeting sequences include, but are not limited to: aheparin binding domain (e.g., polyK, polyR, or combinations thereof); anintegrin binding domain (e.g., RGD, LDV, and the like); a lamininreceptor domain (e.g., YIGSR [SEQ ID NO:66]); a DNA binding domain(e.g., polyK, polyR, or combinations thereof); antibody epitopes (e.g.,the FLAG peptide DYKDDDDK [SEQ ID NO:2] or other epitope); abrain-specific targeting domain (e.g., SLR); and any other peptidedomain which binds to a receptor (e.g., in particular, a peptide domainranging from about 2 to 200 amino acids).

[0089] Along these lines, the method can be employed to increase theefficiency of adenoviral-mediated delivery to, for instance, bone marrowcells, endothelium, organs such as lung, liver, spleen, kidneys, brain,eye, heart, muscle, and the like, hematopoietic cells, tumorvasculature, and tumor cells. Diseases, disorders, or conditionsassociated with these tissues include, but are not limited toangiogenesis, restenosis, inflammation, cancers, Alzheimer's disease,human immunodeficiency virus (HIV-1, HIV-2) infection, and anemias.

[0090] These aforementioned illustrative uses are by no meanscomprehensive, and it is intended that the present invention encompassessuch further uses which flow from, but are not explicitly recited in thedisclosure herein. Similarly, there are numerous advantages associatedwith the use of the various aspects of the present invention.

[0091] For instance, with incorporation of antibody epitopes into thefiber protein, if the antibody epitope is in a loop close to the fiberreceptor binding domain, then binding of the bispecific antibody willblock normal receptor binding, thereby increasing the specificity ofcell targeting using the antibody epitope. If the fiber receptor bindingdomain is mutated such that it no longer binds its receptor, thenincorporation of specific receptor binding domains into the loop willallow targeting to those tissues that express the complementary receptorin the absence of any competing binding mediated by the wild-type fiberreceptor binding domain.

[0092] Similarly, a domain which permits inactivation of fiber for itsnormal receptor binding also can be incorporated into an exposed loop ofthe fiber protein. Inactivation of the fiber binding to its normalreceptor will permit specific targeting via another protein or domain ofadenovirus. For instance, α_(v) integrin targeting with native pentonbase can be accomplished in this fashion. Along these lines, anenterokinase cleavage site (e.g., DYKDDDDK [SEQ ID NO:2]) or trypsincleavage site (e.g., RKKKRKKK [SEQ ID NO:1]) can be incorporated into afiber loop followed by treatment of adenoviral particles withenterokinase or trypsin. Native adenovirus particles are immune to suchenterokinase or trypsin treatment.

[0093] Furthermore, a vector according to the invention, particularly anadenoviral vector, is advantageous in that it can be isolated andpurified by conventional means. Since changes in the vector are made atthe genome level, there are no cumbersome and costly post-productionmodifications required, as are associated with other vectors (see, e.g.,Cotten et al., Proc. Natl. Acad. Sci., 89, 6094-6098 (1992); Wagner etal., Proc. Natl. Acad. Sci., 89, 6099-6103 (1992)). Similarly, specialadenoviral receptor-expressing cell lines are not required. Anadenoviral vector comprising the chimeric fiber protein can bepropagated to similar titers as a wild-type vector lacking the fibermodification.

[0094] Means of Administration

[0095] The vectors and transfer vectors of the present invention can beemployed to contact cells either in vitro or in vivo. According to theinvention “contacting” comprises any means by which a vector isintroduced intracellularly; the method is not dependent on anyparticular means of introduction and is not to be so construed. Means ofintroduction are well known to those skilled in the art, and also areexemplified herein.

[0096] Accordingly, introduction can be effected, for instance, eitherin vitro (e.g., in an ex vivo type method of gene therapy or in tissueculture studies) or in vivo by electroporation, transformation,transduction, conjugation or triparental mating, (co-)transfection,(co-)infection, membrane fusion with cationic lipids, high velocitybombardment with DNA-coated microprojectiles, incubation with calciumphosphate-DNA precipitate, direct microinjection into single cells, andthe like. Similarly, the vectors can be introduced by means of cationiclipids, e.g., liposomes. Such liposomes are commercially available(e.g., Lipofectin, Lipofectamine™, and the like, supplied by LifeTechnologies, Gibco BRL, Gaithersburg, Md.). Moreover, liposomes havingincreased transfer capacity and/or reduced toxicity in vivo (see, e.g.,International Patent Application WO 95/21259) can be employed in thepresent invention. Other methods also are available and are known tothose skilled in the art.

[0097] According to the invention, a “host” (and thus a “cell” from ahost) encompasses any host into which a vector of the invention can beintroduced, and thus encompasses an animal, including, but not limitedto, an amphibian, bird, fish, insect, reptile, or mammal. Optimally ahost is a mammal, for instance, rodent, primate (such as chimpanzee,monkey, ape, gorilla, orangutan, or gibbon), feline, canine, ungulate(such as ruminant or swine), as well as, in particular, human. Desirablysuch a host cell is one in which an adenovirus can exist for a period oftime (i.e., typically from anywhere up to, and potentially even after,about two months) after entry into the cell.

[0098] A cell can be present as a single entity, or can be part of alarger collection of cells. Such a “larger collection of cells” cancomprise, for instance, a cell culture (either mixed or pure), a tissue(e.g., epithelial or other tissue), an organ (e.g., heart, lung, liver,gallbladder, urinary bladder, eye, and other organs), an organ system(e.g., circulatory system, respiratory system, gastrointestinal system,urinary system, nervous system, integumentary system or other organsystem), or an organism (e.g., a bird, mammal, or the like). Preferably,the peptide binding motif employed for cell targeting is such that theorgans/tissues/cells being targeted are of the circulatory system (e.g.,including, but not limited to heart, blood vessels, and blood),respiratory system (e.g., nose, pharynx, larynx, trachea, bronchi,bronchioles, lungs, and the like), gastrointestinal system (e.g.,including mouth, pharynx, esophagus, stomach, intestines, salivaryglands, pancreas, liver, gallbladder, and others), urinary system (e.g.,such as kidneys, ureters, urinary bladder, urethra, and the like),nervous system (e.g., including, but not limited to brain and spinalcord, and special sense organs such as the eye) and integumentary system(e.g., skin). Even more preferably, the cells being targeted areselected from the group consisting of heart, hematopoietic, lung, liver,spleen, kidney, brain, eye, bone marrow, endothelial, muscle, tumorvasculature, and tumor cells.

[0099] One skilled in the art will appreciate that suitable methods ofadministering a vector (particularly an adenoviral vector) of thepresent invention to an animal for purposes of gene therapy (see, forexample, Rosenfeld et al., Science, 252, 431-434 (1991); Jaffe et al.,Clin. Res., 39(2), 302A (1991); Rosenfeld et al., Clin. Res., 39(2),311A (1991); Berkner, BioTechniques, 6, 616-629 (1988); Crystal et al.,Human Gene Ther., 6, 643-666 (1995); Crystal et al., Human Gene Ther.,6, 667-703 (1995)), chemotherapy, and vaccination are available, and,although more than one route can be used for administration, aparticular route can provide a more immediate and more effectivereaction than another route. Pharmaceutically acceptable excipients alsoare well-known to those who are skilled in the art, and are readilyavailable. The choice of excipient will be determined in part by theparticular method used to administer the recombinant vector.Accordingly, there is a wide variety of suitable formulations for use inthe context of the present invention. The following methods andexcipients are merely exemplary and are in no way limiting.

[0100] Moreover, to optimize the ability of the adenovirus to enter thecell by the method of the invention, preferably the method is carriedout in the absence of neutralizing antibodies directed against theparticular adenovirus being introduced intracellularly. In the absenceof such antibodies, there is no possibility of the adenovirus beingbound by the antibody, and thus impeded from binding and/or entering thecell. It is well within the ordinary skill of one in the art to test forthe presence of such neutralizing antibodies. Techniques that are knownin the art can be employed to prevent the presence of neutralizingantibodies from impeding effective protein production (see, e.g.,Crompton et al., supra, International Patent Application WO 96/12406).

[0101] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the compound dissolvedin diluents, such as water, saline, or orange juice; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

[0102] A vector or transfer vector of the present invention, alone or incombination with other suitable components, can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as in a nebulizer or an atomizer.

[0103] Formulations suitable for parenteral administration includeaqueous and non-aqueous, isotonic sterile injection solutions, which cancontain anti-oxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

[0104] Additionally, a vector or transfer vector of the presentinvention can be made into suppositories by mixing with a variety ofbases such as emulsifying bases or water-soluble bases.

[0105] Formulations suitable for vaginal administration can be presentedas pessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

[0106] The dose administered to an animal, particularly a human, in thecontext of the present invention will vary with the gene of interest,the composition employed, the method of administration, and theparticular site and organism being treated. However, the dose should besufficient to effect a therapeutic response.

[0107] As previously indicated, a vector or a transfer vector of thepresent invention also has utility in vitro. Such a vector can be usedas a research tool in the study of adenoviral attachment and infectionof cells and in a method of assaying binding site-ligand interaction.Similarly, the chimeric fiber protein comprising a constrained nonnativeamino acid sequence in addition to or in place of a native amino acidsequence can be used in receptor-ligand assays and as adhesion proteinsin vitro or in vivo, for example.

EXAMPLES

[0108] The following examples further illustrate the present inventionand, of course, should not be construed as in any way limiting itsscope.

Example 1

[0109] This example describes the construction of transfer vectorsencoding fiber sequences having insertions of various peptide motifs inan exposed loop of the knob region of the adenovirus fiber protein.

[0110] The fiber proteins of Ad2 and Ad5 both recognize the samereceptor. A parallel evaluation of the protein structure of the fiberknob and its DNA restriction map reveals that the Ad2 fiber knobcontains a unique Spe I restriction site in a region encoding an exposedloop in the protein. The amino acids in this loop are not involved inany interactions relevant to protein folding. Accordingly, additions tothis loop are highly unlikely to affect the ability of the fiber proteinto fold. Chimeric adenoviral fiber proteins comprising modifications ofan exposed loop (particularly the HI loop) were constructed as describedherein.

[0111] For vector construction and characterization, standard molecularand genetic techniques, such as the generation of strains, plasmids, andviruses, gel electrophoresis, DNA manipulations including plasmidisolation, DNA cloning and sequencing, Western blot assays, and thelike, were performed such as are known to those skilled in the art, andas are described in detail in standard laboratory manuals (e.g.,Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (ColdSpring Harbor, N.Y., 1992); Ausubel et al., Current Protocols inMolecular Biology (1987)). Restriction enzymes and other enzymes usedfor molecular manipulations were purchased from commercial sources(e.g., Boehringer Mannheim, Inc., Indianapolis, Ind.; New EnglandBiolabs, Beverly, Mass.; Bethesda Research Laboratories, Bethesda, Md.),and were used according to the recommendations of the manufacturer.Cells employed for experiments (e.g., cells of the transformed humanembryonic kidney cell line 293 (i.e., CRL 1573 cells) and other cellssupplied by American Type Culture Collection) were cultured andmaintained using standard sterile culture reagents, media andtechniques, as previously described (Erzerum et al., Nucleic AcidsResearch, 21, 1607-1612 (1993)).

[0112] In order to make recombinant adenovirus vectors containingtargeting sequences, it was first necessary to exchange the knob regionof the Ad5 present in a transfer vector with the knob coding region fromAd2, since the HI loop of Ad2 comprises a unique Spe I restriction site,which allows cloning of particular targeting sequences into this site.Accordingly, the net result of the vector manipulations was to create afiber chimera in which the DNA encoding the tail and shaft of the fiberare from Ad5, the. DNA encoding the knob is from Ad2, and the knobfurther comprises a nonnative amino acid sequence in the HI loop asdepicted in FIG. 1. In an alternate method of the invention described inlater Examples, the targeting sequence is placed at the terminus of thefiber knob protein, as depicted in FIG. 2.

[0113] In the first step of the process of making fiber knob insertionsin a loop, the transfer vector p193 (F5*) depicted in FIG. 3 wasconstructed. This plasmid contains an 8 nucleotide insertion between thelast amino acid codon of the fiber coding sequence and the stop codon.The 8 nucleotide insertion contains a unique Bam HI restriction sitewhich allows a straightforward replacement of Ad5 fiber domains withother fiber domains from other adenovirus serotypes. Namely, thesequence of the wild-type Ad5 fiber gene is indicated in SEQ ID NOs:6and 7. In comparison, the C-terminus of the mutated fiber gene presentin p193(F5*) is set forth in SEQ ID NOs: 8 and 9:

[0114] The transfer plasmid p193(F5*) was constructed from p193NS(AF).The mutated fiber gene (i.e., the fiber gene comprising the Bam HI siteprior to the stop codon) was incorporated into the fiber-minus plasmidp193NS(AF) using synthetic sense and antisense oligonucleotide primersto amplify the fiber gene by means of the polymerase chain reaction(PCR) while at the same time incorporating a modified Bam HI sitefollowing the last codon of the fiber gene to create the mutant fibergene. The primers used to amplify from the Nde I site to the C-terminalcoding regions of the fiber gene from Ad5 genome DNA were: antisenseprimer, T CCC CCC GGG TCT AGA TTA GGA TCC TTC TTG GGC AAT GTA TGA (BamHI site underlined) [SEQ ID NO:10]; sense primer CGT GTA TCC ATA TGA CACAGA (Nde I site underlined) [SEQ ID NO:11]. The PCR product was then cutwith Nde I and Bam HI and cloned into the Nde I/Bam HI sites ofp193NS(AF).

[0115] The plasmid p193NS(AF) itself was constructed by means of anintermediary series of vectors. Namely, first, the transfer plasmidp193NS83-100 was constructed by cloning the Ad5 Nde I to Sal I fragment,which spans the 83-100 map unit region of the Ad5 genome containing thefiber gene, into the plasmid pNEB 193 (New England Biolabs, Beverly,Mass.). The Nde I-Mun I fragment was replaced with a syntheticoligonucleotide comprising a Bam HI site, which was flanked by a 5′ NdeI site and a 3′ Mun I site to facilitate cloning. The double-strandedsynthetic oligonucleotide fragment was created from the overlappingsynthetic single-stranded sense (comprising SEQ ID NO:12) and antisense(comprising SEQ ID NO:13) oligonucleotide. The ends of the overlappingoligomers were made to have overhangs compatible for direct cloning intothe Nde I and Mun I sites. The resultant vector p193NS(AF) lacks all thecoding sequence for the fiber gene but contains the entire adenovirus E4coding sequence. The plasmid retains the AATAAA polyadenylation signalincluded in the synthetic Nde I/Mun I oligonucleotide and alsoincorporates the new Bam HI restriction site (underlined).

[0116] Thus, following its construction in a series of sequentialcloning steps, the transfer vector p193(F5*) was employed in subsequentvector constructions. Namely, the sense oligonucleotide FSF2K(s)N(comprising SEQ ID NO:14 and containing a Nco I restriction site) andthe antisense oligonucleotide primer F5F2K(a)B (i.e., comprising SEQ IDNO:15 and containing a Bam HI restriction site) were used to amplify theknob coding region from purified Ad2 DNA by means of PCR. Theincorporation of these sites on either end of the PCR product permittedit to be cut with Nco I and Bam HI and cloned into the base plasmidp193(F5*) to create the transfer vector p193 F5F2K depicted in FIG. 4.Unlike p193(F5*), p193 F5F2K contains a unique Spe I restriction sitewithin the Ad2 fiber gene encoding an exposed loop in the protein.Namely, the fiber gene present in p 193 F5F2K comprises the mutatedfiber sequence (SEQ ID NOs: 16 and 17) including a novel Spe I siteintroduced into the fiber gene This vector was then used to clonetargeting sequences into the Spe I site. In particular, a nucleicsequence encoding the FLAG peptide motif [SEQ ID NO:2] and a nucleicacid sequence encoding the stretch of 8 basic amino acids [SEQ ID NO:1]comprising the heparin binding domain were cloned into the Spe I site ofp193 F5F2K using overlapping sense and antisense oligonucleotides.

[0117] The PolyGS(RKKK)₂ sequence comprises SEQ ID NOs: 18 and 19. The27-mer sense oligonucleotide PolyGS(RKKK)₂(S) (Comprising SEQ ID NO:20)and 27-mer antisense oligonucleotide PolyGS(RKKK)₂(a) (comprising SEQ IDNO:21) were employed for cloning the PolyGS(RKKK)₂ sequence. Thisplasmid was constructed by cloning the DNA sequence encoding the bindingdomain into the Spe I site of p193 F5FK2. The overlapping sense andantisense oligonucleotides encoding the binding domain were firstannealed and then directly ligated into the Spe I restriction site toresult in the plasmid p193 F5F2K(RKKK2) depicted in FIG. 5.

[0118] Similarly, the FLAG sequence comprises SEQ ID NO2: 22 and 23. The30-mer sense oligonucleotide FLAG(s) (comprising SEQ ID NO:24) and30-mer antisense oligonucleotide FLAG(a) (comprising SEQ ID NO:25) wereemployed for cloning the FLAG peptide sequence in a similar fashion asfor p193 F5F2K(RKKK2) to result in the plasmid p193 F5F2K(FLAG) depictedin FIG. 6.

[0119] The FLAG sequence is recognized by the anti-FLAG M2 antibody(Kodak, New Haven, CT) and is used for targeting adenovirus by means ofbispecific antibodies (Wickham et al., “Targeted Adenovirus GeneTransfer to Endothelial and Smooth Muscle Cells Using BispecificAntibodies”, J. Virol., 70(10), in press (1996)). The RKKKRKKK peptidesequence recognizes cellular heparin sulfate and is used to target theadenovirus to heparin sulfate-containing receptors on cells. Becauseheparin sulfate moieties are expressed on nearly all mammalian cells,the heparin-binding motif permits AdF2K(RKKK2) to bind to and transducea broad spectrum of cells, as compared to unmodified (i.e., wild-type)adenovirus vectors.

[0120] The plasmids, p193 F5F2K(RKKK2) and p193 F5F2K(FLAG) wereconfirmed to contain the correct inserts through use of PCR analysis andmobility shift assays done on DNA fragments generated by restrictiondigests of the plasmids. Namely, the relevant portion of the modifiedloop of the fiber knob present in p193 F5F2K(RKKK2) is set forth in SEQID NOs 26 and 27. The relevant portion of the modified loop of the fiberknob present in p193 F5F2K(FLAG) is set forth at SEQ ID NOs: 28 and 29.

[0121] These results thus confirm that the methods described herein canbe employed to construct transfer vectors encoding fiber sequenceshaving insertions of various peptide motifs in an exposed loop of theknob region of the adenovirus fiber protein.

Example 2

[0122] This example describes the construction of adenoviral vectorsencoding fiber sequences having insertions of various peptide motifs ina loop of the knob region of the adenovirus fiber protein.

[0123] The transfer plasmids p193 F5F2K(RKKK2) and p193 F5F2K(FLAG) wereemployed to obtain the corresponding adenoviral vectors comprising theFLAG and RKKK2 peptide motifs. This was accomplished by digesting theseplasmids (which contain the essential E4 region of adenovirus) with SalI, and transfecting them into 293 cells that already had been infected 1hour earlier with the adenovirus vector AdZ.E4Gus. This adenovirusvector lacks the E4 region and cannot replicate in 293 cells without theE4 genes. Only when AdZ.E4Gus DNA recombines with plasmid DNA such asp193 F5F2K, p193 F5F2K(FLAG), and p193 F5F2K(RKKK2) to obtain the E4genes is the vector able to replicate in 293 cells. During thisrecombination to rescue the adenoviral vector, the newly formed vectoralso picks up the mutated fiber sequence encoded by the plasmids.

[0124] Viable recombinant E4⁺ adenovirus containing the F2K(RKKK2) andF2K(FLAG) DNA sequences (i.e., ADZ.FLAG and AdZ.RKKK2) were isolated byplaquing the transfected cell lysates 5 days after transfection. Therecombinant adenoviruses were then plaque-purified 2 times on 293 cells.The purified plaques were amplified on 293 cells. All viruses werepurified from infected cells at 2 days post-infection by 3 freeze-thawcycles followed by two successive bandings on CsCl gradients. Purifiedvirus was dialyzed into 10 mM Tris, 150 mM NaCl, pH 7.8, containing 10mM MgCl₂, 3% sucrose, and was frozen at −80 until required for use. Thepurified viruses were verified by PCR to contain either the RKKK2 insertor the FLAG insert.

[0125] These adenoviral vectors and the sequences they specificallytarget due to their possession of modified fiber knobs are depicted inTable 1. TABLE 1 Adenoviral Vectors Comprising Constrained PeptideMotifs Vector Name Target Receptor Target Sequence AdZ.FLAG Any receptor(with use of a TRDYKDDDDKTS bispecific antibody) [SEQ ID NO:23]AdZ.RKKK2 Heparin sulfate-containing TRKKKRKKKTS receptors [SEQ ID NO:19]

[0126] These results thus confirm that the methods described herein canbe employed to construct adenoviral vectors encoding fiber sequenceshaving insertions of various peptide motifs in an exposed loop of theknob region of the adenovirus fiber protein.

Example 3

[0127] This example describes the characterization of adenoviral vectorsencoding fiber sequences having insertions of various peptide motifs ina loop of the knob region of the adenovirus fiber protein.

[0128] The FLAG insert present in the ADZ.FLAG vector was shown to befunctionally accessible and capable of binding the anti-FLAG M2 mAB asassessed by immunofluorescence, as previously described (Wickham et al.,1993). Briefly, 293 cells were infected at a low multiplicity ofinfection (i.e., about a 0.02 MOI) with the AdZ.RKKK2 or ADZ.FLAGisolates. The cells were fixed at two days post-infection and incubatedwith either a rabbit anti-penton base polyclonal antibody or a mouseanti-FLAG mAB, followed by incubation with anti-rabbit or anti-mouseFITC antibody. The anti-penton base antibody recognized cells infectedby either virus. In comparison, the FLAG mAB recognized only the cellsinfected with the ADZ.FLAG virus, and not the cells infected with theAdZ.RKKK2 virus.

[0129] These results confirm that adenoviruses produced according to themethod of the invention are viable, and that the insert (e.g., FLAGepitope) present in an exposed loop of fiber protein is accessible toand capable of binding its corresponding binding entity (e.g., a cellsurface binding site or an antibody such as the anti-FLAG antibody).These results confirm that the method of the invention can be employedfor adenoviral-mediated cell targeting.

Example 4

[0130] This example describes gene delivery mediated by adenoviralvectors encoding fiber sequences having insertions of various peptidemotifs in an exposed loop of the knob region of the adenovirus fiberprotein.

[0131] For testing the ability of the RKKK2 motif to effect celltargeting, 293 cells (which appear to express relatively high levels ofthe receptor by which wild-type adenovirus fiber protein effects cellentry) were preincubated for 30 minutes in the presence and absence ofcompeting wild-type fiber protein. Purified AdZ or AdZ.RKKK2 vectorswere then incubated with the cells for an additional 60 minutes at 37 C.The cells were washed 3 times with PBS, and incubated in culture mediumovernight. β-galactosidase activity from lysed cells was then determinedusing a β-galactosidase fluorometric assay kit (Tropix, Bedford, Mass.).Activity was measured in a luminometer in relative light units (RLU).

[0132] The data illustrated in FIG. 7 demonstrates gene delivery to 293cells effected by the AdZ.RKKK2 vector. As can be seen from this figure,recombinant wild-type fiber protein blocked gene delivery by AdZ, butnot by AdZ.RKKK2. The AdZ.RKKK2 vector was able to overcome thefiber-mediated block to adenoviral-mediated gene delivery.

[0133] These results confirm that this constrained peptide motif presentin the fiber loop is able to efficiently mediate cell binding/entry.Moreover, the results further confirm that adenoviral vectors encodingfiber sequences having insertions of various peptide motifs in anexposed loop of the knob of the adenovirus fiber protein can be employedfor delivery (e.g., of DNA and/or protein) to cells.

Example 5

[0134] This example describes other oligonucleotides that can beemployed for inserting a nonnative amino acid sequence into a chimericadenovirus fiber protein, preferably in an exposed loop of theadenovirus fiber knob, but also at the C-terminus of the protein.

[0135] The cloning techniques described in the previous example can beemployed to incorporate into an exposed loop of the fiber knob insertscomprising peptide motifs that will target, for instance, α_(v)integrins, α₅β₁ integrin, FLAG mAb, or other cell surface binding sites.In particular, an HAαv sequence (e.g., comprising SEQ ID NOs: 30 and 31)can be inserted. For example, the sequence can be inserted with use ofthe 39-mer sense oligonucleotides HAav(s) (comprising SEQ ID NO:32) andHAαv(a) (comprising SEQ ID NO:33). These oligonucleotides were used tomake p193(F5*)pGS(RGD), which was used to make AdZ.RGD.

[0136] Similarly, an HAα5β1 sequence (e.g., SEQ ID NOs: 34 and 35) canbe inserted for targeting integrin α5β1. The sequence can be insertedwith use of the 39-mer sense oligonucleotides HAα5β1 (s) (comprising SEQID NO:36) and HAα5β1(a) (comprising SEQ ID NO:37).

[0137] These sequences (and other sequences described herein) that allowtargeting to the α_(v) integrins are of use since this target receptordemonstrates broad distribution, including to endothelial cells andsmooth muscle cells. The adhesion receptor appears to be important inwounds (i.e., both healing and exacerbation thereof), as well as inangiogenesis, restenosis and metastasis. Generally, the receptor isupregulated in proliferating endothelial cells and smooth muscle cells,and exhibits high expression in melanoma and glioma. Normal ligands forthe α_(v) integrins receptor include vitronectin, collagen, fibronectin,laminin, and osteopontin.

[0138] Also, an E-selectin targeting sequence (e.g., comprising SEQ IDNOs: 38 and 39) can be inserted. The E-selectin sequence can be insertedwith use of the 42-mer sense oligonucleotide E-selectin(s) (comprisingSEQ ID NO:40) and the 42-mer antisense oligonucleotide E-selectin(a)(comprising SEQ ID NO:41). Further ligands that bind elastin have beendescribed in the art and similarly can be employed as nonnative aminoacid sequences for the generation of peptide motifs as described herein(see, e.g., Martens et al., J. Biolog. Chem., 270, 21129-21136 (1995)).

[0139] Furthermore, a PolyGS(RKKK)3 sequence (e.g., SEQ ID NOs: 42 and43), or other variations of this sequence, can be inserted. The sequencecan be inserted with use of the 39-mer sense oligonucleotidePolyGS(RKKK)₃(s) (comprising SEQ ID NO:44) and the 39-mer antisenseoligonucleotide PolyGS(RKKK)₃(a) (comprising SEQ ID NO:45).

[0140] This example thus confirms that other oligonucleotides can beemployed for inserting a nonnative amino acid sequence into a fiberprotein. Such insertions can either be made in an exposed loop of theadenovirus fiber knob or, as described as follows, at the C-terminus ofthe fiber protein. Moreover, the nonnative amino acid sequence can beincorporated into the chimeric fiber protein not merely as an insertioninto the sequence, but also as a replacement of adenoviral sequences.This can be done through modification of the cloning proceduresdescribed herein, as are known to those skilled in the art.

Example 6

[0141] In a similar fashion to the constraint achieved by placing apeptide motif within an exposed loop of the adenovirus fiber protein,constraint can be obtained through appropriate modification of a peptidemotif at the C-terminus of the fiber protein to create, in essence, anonpreexisting loop at this site. Thus, this example describes theconstruction of transfer vectors encoding fiber sequences havinginsertions of various constrained peptide motifs at the C-terminus ofthe adenovirus fiber protein. This method is depicted in FIG. 2.

[0142] The transfer vector p193(F5*) described in Example 1 was used asa base plasmid to create chimeric adenovirus particles containingC-terminal additions to the fiber gene. In particular, DNA sequencesencoding a linker sequence followed by a targeting sequence and a stopcodon were cloned into the Bam HI site to create further transfervectors which, in turn (i.e., via the construction of the furthertransfer vectors p193(F5)pGS(RGD) and p193(F5)pGS) were used to makechimeric adenovirus particles.

[0143] The mutant transfer plasmids containing sequences encoding anamino acid glycine/serine repeat linker, a targeting sequence, and astop codon were made by cloning synthetic oligonucleotides into the BamHI site ofp193(F5*). The cloning reactions essentially were carried outas described in Example 1. In particular, the overlapping syntheticoligonucleotides used to make the transfer plasmid p193(F5)pGS(RGD)depicted in FIG. 8 were: sense, SEQ ID NO:46; antisense, SEQ ID NO:47.This plasmid comprises SEQ ID NO:48, encoding the amino acid sequenceSEQ ID NO:49. The RGD peptide is present within this larger sequence.The plasmid p193(F5)pGS(RGD) thus comprises the targeting sequenceCDCRGDCFC [SEQ ID NO:3] which is present in the larger sequence SEQ IDNO:86. This sequence, like other sequences described earlier containingthe tripeptide motif RGD, acts as a ligand for the target receptor a5integrins. However, highly constrained forms of RGD bind with higheraffinities to integrins than linear forms (see, e.g., Aumailley et al.,FEBS, 291, 50-54 (1991); Cardarelli et al., J. Biolog. Chem., 269.,18668-18673 (1994); Koivunen et al., Bio/Technology, 13, 265-270(1995)). Along these lines, the constrained RGD targeting motif presentin p193(F5)pGS(RGD) binds with about 100-fold higher affinity to α_(v)integrins than does similar linear RGD motifs. Each pair of cysteines oneither side of the RGD form disulfide binds with the opposite pair ofcysteines to form a highly constrained RGD loop.

[0144] Moreover, variations of the CDCRGDCFC [SEQ ID NO:3] targetingsequence can be employed in the context of the present invention. Forinstance, instead of two cysteine residues on either side of the RGDtripeptide sequence, only one residue can be used instead. Any sequencecan be employed, so long as a loop-like structure is createdencompassing the RGD sequence, and so long as the sequence comprises oneor more cysteine pairs. Moreover, the RGD sequence can be substituted byanother sequence, e.g., LDV.

[0145] In terms of construction of the related transfer plasmidp193(F5)pGS, the overlapping synthetic oligonucleotides used to make thetransfer plasmid were: sense, PolyGS(s), [SEQ ID NO:50]; antisense,PolyGS (a), [SEQ ID NO:51]. The sense and antisense oligonucleotideswere mixed in equimolar ratios and cloned into the Bam HI siteofp193(F5*) to create p193(F5)pGS. The transfer vectorp193(F5)pGS thenwas used to construct further transfer vectors, as described in thefollowing Examples.

[0146] Thus, this example confirms that transfer vectors encoding fibersequences having insertions of various constrained peptide motifs at theC-terminus of the adenovirus fiber protein can be constructed accordingto the invention. Other transfer vectors (i.e., having differenttargeting sequences) also can be constructed using this approach.

Example 7

[0147] This example describes the construction of adenovirus vectorsencoding fiber sequences having insertions of various constrainedpeptide motifs at the C-terminus of the adenovirus fiber protein.

[0148] The E1- and E3-deleted adenovirus AdZ employed for theseexperiments contains the β-galactosidase gene under the control of acytomegalovirus (CMV) promoter and integrated into the adenoviralgenome. AdZ was propagated in human embryonic kidney 293 cells, whichcontain the complementary E1 region for virus growth. AdZ.RGD (as wellas other vectors targeted to other adhesion receptors described herein)was derived directly from AdZ. These viruses likewise are E1- andE3-deleted, and are identical to AdZ, except for the presence ofadditional amino acids on the C-terminus of the fiber proteins.

[0149] The transfer plasmids, p193(F5)pGS and p193(F5)pGS(RGD), whichcontain the essential E4 region of adenovirus, were employed foradenoviral vector construction. These transfer plasmids were cut withSal I and transfected into 293 cells that had been infected one hourprior with the adenovirus vector, AdZ.E4Gus. The adenovirus vectorAdZ.E4Gus lacks the E4 region and cannot replicate in 293 cells withoutthe E4 genes. Only when AdZ.E4Gus DNA recombines with the p193(F5)pGS orp193(F5)pGS(RGD) plasmid DNA to obtain the E4 genes is the vector ableto replicate in 293 cells. During this recombination, the newly formedvector also picks up the fiber mutations encoded in the plasmids. Viablerecombinant E4+adenovirus containing the pGS and pGS(RGD) mutations werethen isolated by plaquing the transfected cell lysates 5 days aftertransfection. Their resultant vectors, AdZ.pGS and ADZ.RGD, wereisolated and purified by two successive rounds of plaquing on 293 cells.Each vector was verified to contain the correct insert by sequencing PCRproducts from virus DNA that spans the region of the insert DNA.

[0150] This example confirms that adenovirus vectors encoding fibersequences having insertions of various constrained peptide motifs at theC-terminus of the adenovirus fiber protein can be constructed accordingto the invention.

Example 8

[0151] This example describes the construction of transfer vectors andadenoviral vectors with use of other oligonucleotides that can beemployed for inserting a normative amino acid sequence into a chimericadenovirus fiber protein, preferably in an exposed loop of theadenovirus fiber knob, but also at the C-terminus of the protein.

[0152] The cloning techniques described in Example 6 were employed tocreate additions at the C-terminus. Basically the transfer vectorsdescribed in this Example (in particular, the transfer vectorp193(F5)pGS) were linearized at the unique cloning site Spe I present inthe vectors, and new sequences were inserted at this site. Other means(e.g., PCR reactions) also can be employed to make insertions into thisunique site. Similarly, the cloning techniques described in Example 5can be employed to incorporate into an exposed loop of the fiber knobinserts comprising peptide motifs that target other cell surface bindingsites or epitopes for an antibody.

[0153] In particular, multiple copies of the RGD sequence (i.e., apolyRGD or pRGD sequence; SEQ ID NOs: 52 and 53) were inserted. Thesequence was inserted with use of the sense oligonucleotide pRGDs(comprising SEQ ID NO:54) and the antisense oligonucleotide pRGDa(comprising SEQ ID NO:55). The resultant plasmid p193(F5*)RGD wasemployed to create the adenovirus AdZ.pRGD. A comparison of the insertspresent in ADZ.RGD and AdZ.pRGD (with the RGD peptide indicatedemboldened) is presented in Table 2. TABLE 2 Comparison of AdenoviralVectors AdZ.RGD and AdZ.pRGD Vector Name Target Receptor Target SequenceAdZ.RGD α_(v) Integrins SACDCRGDCFCGTS [SEQ ID NO:68] AdZ.pRGD α_(v)Integrins TS(GRGDTF)₃SS β₁ Integrins [SEQ ID NO:52]

[0154] Similarly, one or more copies of an LDV targeting sequence can beinserted. The LDV target receptor is distributed in hematopoietic cells,lymphocytes, and monocytes/macrophages. The adhesion receptor is highlyexpressed on resting lymphocytes involved in cell-matrix and cell-cellinteractions (e.g., during hematopoietic extravasation, as well asinflammation, and lymphocyte trafficking). Ligands for the α₄ integrinstarget receptor include, but are not limited to, fibronectin (anextracellular matrix protein), VCAM-1 (which targets endothelialtissue), and MAdCAM (α₄β₇)(which is gut-specific). In particular, the α₄integrins targeting sequences includes the sequence EILDVPST ([SEQ IDNO:56] encompassed by the sequence above, and the sequence (EILDVPS)₃[SEQ ID NO:57].

[0155] In particular, multiple copies of the LDV sequence (i.e., apolyLDV or pLDV sequence) can be inserted to comprise SEQ ID NOs:58 and59. This sequence was inserted with use of the sense oligonucleotidepLDVs (comprising SEQ ID NO:60) and the antisense oligonucleotide pLDVa(comprising the sequence SEQ ID NO:61).

[0156] Such insertion resulted in the generation of the vectorp193(F5)pLDV depicted in FIG. 9. The LDV targeting motif present in thisvector (i.e., comprising the sequence of SEQ ID NO:59) binds withsub-millimolar affinity to α₄ integrins. The LDV motif is repeated 3times in each fiber monomer for a total of 9 motifs per fiber molecule.This vector further was employed for the generation of a correspondingadenoviral vector.

[0157] Furthermore, a pYIGSR targeting sequence was inserted at theC-terminus of the fiber protein to derive the plasmid p193(F5)pYIGSRdepicted in FIG. 10. The fiber protein in this plasmid comprises SEQ IDNOs: 62 and 63. The sequence was inserted with use of the senseoligonucleotide pYIGSRs (comprising SEQ ID NO:64) and the antisenseoligonucleotide pYIGSRa (comprising SEQ ID NO:65).

[0158] The resultant plasmid contains the YIGSR [SEQ ID NO:66] targetingmotif, which binds with sub-millimolar affinity to the high affinitylaminin receptor. The motif, present as YIGSRG (SEQ ID NO:67), isrepeated 3 times in each fiber monomer for a total of 9 motifs per fibermolecule. In particular, the motif provides for targeting to the 67kilodalton laminin/elastin receptor. This receptor is present inmonocytes/neutrophils, vascular smooth muscle, fibroblasts, andchondrocytes, and is upregulated in multiple tumors. Furthermore, thereceptor appears to be involved in tumor metastasis and angiogenesis.Typical ligands for the laminin/elastin receptor include laminin,elastin, and galactose. The p193(F5)pYIGSR plasmid derived hereinfurther was employed to create the adenovirus vector AdZ.pYIGSR.

[0159] This example thus confirms that other oligonucleotides can beemployed for inserting a nonnative amino acid sequence into a fiberprotein. Such insertions can eitherbe made in an exposed loop of theadenovirus fiber knob, or, as described as follows, at the C-terminus ofthe fiber protein. Moreover, the nonnative amino acid sequence can beincorporated into the chimeric fiber protein not merely as an insertioninto the sequence, but also, as a replacement of adenoviral sequences.This can be done through simple modification of the cloning proceduresdescribed herein, such as are known to those skilled in the art.

Example 9

[0160] This example describes the characterization of adenoviral vectorsencoding fiber sequences having an insertion of a constrained RGDpeptide motif at the C-terminus of the adenovirus fiber protein. Inparticular, the ability of these vectors to produce active virusparticles in different cells was investigated.

[0161] For the Western analysis of virus particles, purified virusparticles (2×10¹⁰) in a volume of 10 μl were diluted 1:1 in Laemmlirunning buffer and loaded onto a 9% acrylamide, 0.1% SDS gel. The gelwas run at 150 mV and was then transferred to nitrocellulose. Thenitrocellulose was blocked with 5% dry milk and probed with acombination of rabbit polyclonal antibodies directed against denaturedAdS virions (1:1000) and against fiber protein (1:5000). The proteinswere detected using antirabbit-peroxidase (1:5000) and a commerciallyavailable chemiluminescent detection kit.

[0162] The fiber proteins of the recombinant adenoviruses AdZ.pGS andADZ.RGD were shifted upward on the Western relative to the fiber proteincontained by the AdZ vector. A gel run in parallel that was transferredto nitrocellulose and probed using only the polyclonal antibody directedagainst the fiber protein demonstrated that the shifted bands in theWestern analysis were, in fact, fiber protein. These results confirmthat the AdZ.pGS and ADZ.RGD fiber proteins contain the appropriateamino acid inserts.

[0163] The viral production kinetics were determined to confirm thatviable adenovirus was being produced in 293 cells infected with variousadenoviral vectors according to the invention. To carry out thesestudies, radiolabeled adenovirus was made by adding 50 μCi/ml[³H]thymidine (Amersham, Arlington Heights, Ill.) to the medium ofinfected cells at 20 hours following their infection at an MOI of 5. Theinfected cells were then harvested at 60 hours post-infection, and thevirus was purified as previously described. The activity of the labeledviruses was approximately 10⁴ virus particles/cpm. Infectious particleswere titered in fluorescence focus units (ffu) using a fluorescent focusassay on 293 cells.

[0164] Active virus particle production kinetics from infected 293 cellswere determined by infecting 10⁶293 cells with 0.2 ml of either AdZ orAdZ.RGD for 1 hour in 6 cm plates at an MOI of 10 on day 0. The cellswere harvested on 1, 2, and 3 days post-infection. The cells were spundown and resuspended in 1 ml of PBS for AdZ and ADZ.RGD. The cells werefrozen and thawed 3 times to release the virus particles. The lysateswere then assayed for the number of active particles produced per cellusing standard techniques. The results of these experiments (depicted inFIG. 11) confirm that the modifications to the fiber protein in ADZ.RGDdo not significantly affect the production of active virus particlescompared to the unmodified vector, AdZ.

[0165] The particle dose-response of the vectors AdZ and AdZ.RGD on A549epithelial, CPAE endothelial, and human intestinal smooth muscle (HISM)cells similarly was investigated. HISMC, CPAE, or A549 cells (5×10⁵cells/well) were seeded onto 6 cm plates 1-2 days prior to experiments.In assays evaluating the vector dose-response in fiberreceptor-expressing cells, increasing concentrations of AdZ or AdZ.RGDparticles were incubated with the cells for 60 minutes at 37 C in 0.2 mlDMEM+20 mM HEPES. The plates were shaken every 10 minutes during thisincubation. The cells were then washed 2 times with DMEM and cultured inDMEM+5% calf serum for 2-3 days at 37 C. The medium was than aspirated,and the cells were lysed in 1 ml 1× reporter lysis buffer+10 mM EDTA(Promega, Madison, Wis). The β-galactosidase activity in the celllysates was then assayed as previously described. Results are theaverage of duplicate measurements.

[0166] The results of these experiments are presented in FIGS. 12-14.These experiments confirm that the AdZ and AdZ.RGD vectors areequivalent in terms of their ability to enter and produce viable virusparticles in cells (A549) known to express high levels of adenovirusfiber receptor (i.e., A549 cells as presented in FIG. 12). However, forthe CPAE and HISM cells (i.e., presented in FIG. 13 and FIG. 14,respectively) which lack significant levels of adenovirus fiberreceptor, but do express α_(v) integrins, the ADZ.RGD vector is muchmore efficient in transduction than is the unmodified, AdZ vector.Transduction of the CPAE and HISM cells by ADZ.RGD is roughly 100-foldand 30-fold higher, respectively, than AdZ over a wide range of vectorconcentrations.

[0167] These results validate that amino acid inserts present inadenoviral vectors according to the invention are appropriatelytranslated within the context of the chimeric adenovirus fiber protein,and that the resultant chimeric fiber protein is functional, as assessedby the generation of viable adenoviruses containing this protein.Moreover, the results confirm that the peptide motif present in thechimeric fiber protein is able to redirect adenovirus binding, and toselectively effect adenoviral cell binding/entry with a high efficiency.

Example 10

[0168] This example describes the binding behavior of adenoviral vectorsencoding fiber sequences having an insertion of a various constrainedpeptide motif at the C-terminus of the adenovirus fiber protein.

[0169] The specificity of the AdZ and the ADZ.RGD vectors in binding tokidney (835), smooth muscle (A10), and endothelial (CPAE) cells wasstudied. For these experiments, monolayers of 835, A10, or CPAE cells in24 well tissue culture plates were preincubated for 45 minutes with 0.3ml medium containing soluble recombinant fiber (F5; 3 ug/ml), pentonbase (PB; 50 μg/ml), fiber plus penton base, or neither coat protein.Radiolabeled AdZ or ADZ.RGD was then added to the wells and incubatedfor 90 minutes while rocking at room temperature. The wells were washed3 times with PBS, and the remaining cell-associated radioactivity wasdetermined in a scintillation counter. The results of these experimentsare presented graphically in FIGS. 15-17, and quantitatively in Table 3.TABLE 3 Comparison of AdZ and AdZ.RGD binding to three cell lines* 835HEK** CPAE** A10** AdZ AdZ.RGD AdZ AdZ.RGD AdZ AdZ.RGD Control 7.6 12.70.19 0.84 0.72 1.68 Fiber 1.7 12.3 0.22 1.06 0.23 1.40 PB 9.0 9.7 0.200.37 0.80 0.62 Fiber/PB 1.0 3.7 0.21 0.46 0.20 0.41

[0170] These results confirm that fiber protein significantly blocks AdZtransduction, but not ADZ.RGD transduction of both the 835 (FIG. 15) andA10 (FIG. 16) cells. Only fiber plus penton base, which, in combination,blocks both fiber receptor and α_(v) integrins, is able to significantlyblock binding of ADZ.RGD to these cells. For the CPAE cells which lackdetectable levels of fiber receptor (FIG. 17) penton base alone is ableto significantly block binding of ADZ.RGD.

[0171] These results demonstrate that ADZ.RGD interacts with α_(v)integrins on cells. Moreover, the results validate that the peptidemotif as present in the fiber protein of AdZ.RGD can effectively beemployed to target adenovirus to particular cells.

Example 11

[0172] This example describes gene delivery mediated by adenoviralvectors encoding insertions of various sequences at the C-terminus ofthe adenovirus fiber protein.

[0173] For testing the ability of the YIGSR peptide motif to effect celltargeting, A549 cells were preincubated for 30 minutes in the presenceand absence of competing wild-type fiber protein. Purified AdZ orAdZ.pYIGSR vectors were then incubated with the cells for an additional60 minutes at 37 C. The cells were then washed 3 times with PBS andincubated in culture medium overnight. β-galactosidase activity from thelysed cells was determined.

[0174] As presented in FIG. 18, recombinant wild-type fiber proteincompletely blocked gene delivery by both vectors. Increased genedelivery by the AdZ.pYIGSR vector is not observed in the presence offiber protein. This indicates that the pYIGSR targeting motif is not ofsufficiently high affinity to overcome the block to adenovirus bindingthat is achieved with the addition of soluble fiber protein.

[0175] For testing the ability of the pLDV motif to effect celltargeting, Ramos cells (which express high levels of the α₄ integrintarget receptor) were preincubated for 30 minutes in the presence andabsence of competing wild-type fiber protein. The purified AdZ orAdZ.pLDV vectors were then incubated with the cells for an additional 60minutes at 37° C. The cells were washed 3 times with PBS, and incubatedin culture medium overnight. β-galactosidase activity from the lysedcells was then determined.

[0176]FIG. 19 illustrates gene delivery to Ramos cells effected by theAdZ.pLDV vector. As can be seen from this figure, recombinant wild-typefiber protein blocked gene delivery by both AdZ and AdZ.pLDV. As withAdZ.pYIGSR, there is no evidence of increased gene delivery effected bythe AdZ.pLDV vector in the presence of fiber protein. This indicatesthat the pLDV targeting motif, like the YIGSR targeting motif, is not ofsufficiently high affinity to overcome the fiber-mediated block toprotein binding. The remaining gene delivery capacity of AdZ.pLDV thatis not blocked by the addition of soluble fiber protein also is notblocked by further incubation with EDTA. In comparison, the interactionof the α₄ integrins with the LDV motif normally present in fibronectinis blocked by EDTA. This result further confirms that the pLDV targetingmotif is not interacting with high enough affinity with α₄ integrins toincrease vector binding and gene delivery to the Ramos cells. However,with both the YIGSR motif (i.e., comprising the sequence of [SEQ IDNO:66] and the LDV motif, it is possible that high affinity peptidemotifs could be derived by the conformational restraint of thesepeptides in an exposed loop of the fiber proteins.

[0177] The ability of the RGD motif to effect cell targeting similarlywas studied in av-integrins expressing 293 cells. These studies werecarried out as for the other peptide motifs/cell lines. However, forcomparative purposes, the vectors AdZ and AdZ.pRGD (i.e., the vectorcontaining multiple copies of the RGD motif not having cysteineresidues) were also included. The results of these studies are presentedin FIG. 20. As can be seen from this figure, ADZ.RGD, but not AdZ.pRGD,clearly was able to overcome the fiber-mediated block toadenoviral-mediated gene delivery.

[0178] These results thus confirm that the RGD peptide motif (i.e.,present as a loop at the C-terminus of the fiber protein), like theRKKK2 motif present in a loop of the adenovirus fiber protein (describedin Example 4), is of sufficiently high affinity that it was able toovercome the fiber-mediated block to adenoviral-mediated gene delivery,and effectively “swamp out” the typical interaction of wild-type fiberprotein with its cellular receptor to target the adenovirus to a newreceptor.

[0179] The results further confirm that the constraint of a nonnativeamino acid sequence (i.e., either through insertion in a fiber loop orcreation of a loop like structure at the fiber terminus) can result inthe creation of a high affinity peptide motif. Such a high affinitypeptide motif is of use in adenoviral cell targeting.

[0180] All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference to the same extent as if each reference were setforth in its entirety herein.

[0181] While this invention has been described with an emphasis uponpreferred embodiments, it will be apparent to those of ordinary skill inthe art that variations in the preferred embodiments can be prepared andused and that the invention can be practiced otherwise than asspecifically described herein. The present invention is intended toinclude such variations and alternative practices. Accordingly, thisinvention includes all modifications -encompassed within the spirit andscope of the invention as defined by the following claims.

1 80 8 amino acids amino acid single linear peptide 1 Arg Lys Lys LysArg Lys Lys Lys 1 5 8 amino acids amino acid single linear peptide 2 AspTyr Lys Asp Asp Asp Asp Lys 1 5 9 amino acids amino acid single linearpeptide 3 Cys Asp Cys Arg Gly Asp Cys Phe Cys 1 5 9 amino acids aminoacid single linear peptide 4 Cys Xaa Cys Arg Gly Asp Cys Xaa Cys 1 5 23amino acids amino acid single linear peptide 5 Cys Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Cys Arg Gly Asp Cys Xaa Xa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa XaaCys 20 22 base pairs nucleic acid double linear DNA (genomic) 6 TCA TACATT GCC CAA GAA TAA A 22 Ser Tyr Ile Ala Gln Glu 1 5 6 amino acids aminoacid linear peptide 7 Ser Tyr Ile Ala Gln Glu 1 5 30 base pairs nucleicacid double linear DNA (genomic) 8 TCA TAC ATT GCC CAA GAA GGA TCC AATAAA 30 Ser Tyr Ile Ala Gln Glu Gly Ser Asn Lys 10 15 10 amino acidsamino acid linear peptide 9 Ser Tyr Ile Ala Gln Glu Gly Ser Asn Lys 1 510 43 base pairs nucleic acid single linear other nucleic acid 10TCCCCCCGGG TCTAGATTAG GATCCTTCTT GGGCAATGTA TGA 43 21 base pairs nucleicacid single linear other nucleic acid 11 CGTGTATCCA TATGACACAG A 21 55base pairs nucleic acid single linear other nucleic acid 12 TATGGAGGATCCAATAAAGA ATCGTTTGTG TTATGTTTCA ACGTGTTTAT TTTTC 55 57 base pairsnucleic acid single linear other nucleic acid 13 AATTGAAAAA TAAACACGTTGAAACATAAC ACAAACGATT CTTTATTGGA TCCTCCA 57 54 base pairs nucleic acidsingle linear other nucleic acid 14 GGCCATGGCC TAGAATTTGA TTCAAACGGTGCCATGATTA CTAAACTTGG AGCG 54 29 base pairs nucleic acid single linearother nucleic acid 15 GCGGATCCTT ATTCCTGGGC AATGTAGGA 29 36 base pairsnucleic acid double linear DNA (genomic) 16 ATT ACA CTT AAT GGC ACT AGTGAA TCC ACA GAA ACT 36 Ile Thr Leu Asn Gly Thr Ser Glu Ser Thr Glu Thr15 20 12 amino acids amino acid linear peptide 17 Ile Thr Leu Asn GlyThr Ser Glu Ser Thr Glu Thr 1 5 10 33 base pairs nucleic acid doublelinear DNA (genomic) 18 ACT AGA AAA AAA AAA CGC AAG AAG AAG ACT AGT 33Thr Arg Lys Lys Lys Arg Lys Lys Lys Thr Ser 15 20 11 amino acids aminoacid linear peptide 19 Thr Arg Lys Lys Lys Arg Lys Lys Lys Thr Ser 1 510 27 base pairs nucleic acid single linear other nucleic acid 20CTAGAAAGAA GAAACGCAAA AAGAAGA 27 27 base pairs nucleic acid singlelinear other nucleic acid 21 CTAGTCTTCT TTTTGCGTTT CTTCTTT 27 36 basepairs nucleic acid double linear DNA (genomic) 22 ACT AGA GAC TAC AAGGAC GAC GAT GAT AAG ACT AGT 36 Thr Arg Asp Tyr Lys Asp Asp Asp Asp LysThr Ser 15 20 12 amino acids amino acid linear peptide 23 Thr Arg AspTyr Lys Asp Asp Asp Asp Lys Thr Ser 1 5 10 30 base pairs nucleic acidsingle linear other nucleic acid 24 CTAGAGACTA CAAGGACGAC GATGATAAGA 3030 base pairs nucleic acid single linear other nucleic acid 25CTAGTCTTAT CATCGTCGTC CTTGTAGTCT 30 63 base pairs nucleic acid doublelinear DNA (genomic) 26 ATT ACA CTT AAT GGC ACT AGA AAG AAG AAA CGC AAAAAG AAG ACT AGT 48 Ile Thr Leu Asn Gly Thr Arg Lys Lys Lys Arg Lys LysLys Thr Ser 15 20 25 GAA TCC ACA GAA ACT 63 Glu Ser Thr Glu Thr 30 21amino acids amino acid linear peptide 27 Ile Thr Leu Asn Gly Thr Arg LysLys Lys Arg Lys Lys Lys Thr Ser 1 5 10 15 Glu Ser Thr Glu Thr 20 66 basepairs nucleic acid double linear DNA (genomic) 28 ATT ACA CTT AAT GGCACT AGA GAC TAC AAG GAC GAC GAT GAT AAG ACT 48 Ile Thr Leu Asn Gly ThrArg Asp Tyr Lys Asp Asp Asp Asp Lys Thr 25 30 35 AGT GAA TCC ACA GAA ACT66 Ser Glu Ser Thr Glu Thr 40 22 amino acids amino acid linear peptide29 Ile Thr Leu Asn Gly Thr Arg Asp Tyr Lys Asp Asp Asp Asp Lys Thr 1 510 15 Ser Glu Ser Thr Glu Thr 20 45 base pairs nucleic acid doublelinear DNA (genomic) 30 ACT AGA GCC TGC GAC TGT CGC GGC GAT TGT TTT TGCGGT ACT AGT 45 Thr Arg Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly ThrSer 25 30 35 15 amino acids amino acid linear peptide 31 Thr Arg Ala CysAsp Cys Arg Gly Asp Cys Phe Cys Gly Thr Ser 1 5 10 15 39 base pairsnucleic acid single linear other nucleic acid 32 CTAGAGCCTG CGACTGTCGCGGCGATTGTT TTTGCGGTA 39 39 base pairs nucleic acid single linear othernucleic acid 33 CTAGTACCGC AAAAACAATC GCCGCGACAG TCGCAGGCT 39 39 basepairs nucleic acid double linear other nucleic acid 34 ACT AGA TGC CGCCGC GAA ACC GCT TGG GCC TGT ACT AGT 39 Thr Arg Cys Arg Arg Glu Thr AlaTrp Ala Cys Thr Ser 20 25 13 amino acids amino acid linear peptide 35Thr Arg Cys Arg Arg Glu Thr Ala Trp Ala Cys Thr Ser 1 5 10 33 base pairsnucleic acid single linear other nucleic acid 36 CTAGATGCCG CCGCGAAACCGCTTGGGCCT GTA 33 33 base pairs nucleic acid single linear other nucleicacid 37 CTAGTACAGG CCCAAGCGGT TTCGCGGCGG CAT 33 48 base pairs nucleicacid double linear DNA (genomic) 38 ACT AGA GAC ATT ACC TGG GAC CAG CTTTGG GAC CTT ATG AAG ACT AGT 48 Thr Arg Asp Ile Thr Trp Asp Gln Leu TrpAsp Leu Met Lys Thr Ser 15 20 25 16 amino acids amino acid linearpeptide 39 Thr Arg Asp Ile Thr Trp Asp Gln Leu Trp Asp Leu Met Lys ThrSer 1 5 10 15 42 base pairs nucleic acid single linear other nucleicacid 40 CTAGAGACAT TACCTGGGAC CAGCTTTGGG ACCTTATGAA GA 42 42 base pairsnucleic acid single linear other nucleic acid 41 CTAGTCTTCA TAAGGTCCCAAAGCTGGTCC CAGGTAATGT CT 42 45 base pairs nucleic acid double linear DNA(genomic) 42 ACT AGA AAG AAG AAG CGC AAA AAA AAA AGA AAG AAG AAG ACT AGT45 Thr Arg Lys Lys Lys Arg Lys Lys Lys Arg Lys Lys Lys Thr Ser 15 20 2515 amino acids amino acid linear peptide 43 Thr Arg Lys Lys Lys Arg LysLys Lys Arg Lys Lys Lys Thr Ser 1 5 10 15 39 base pairs nucleic acidsingle linear other nucleic acid 44 CTAGAAAGAA GAAGCGCAAA AAAAAAAGAAAGAAGAAGA 39 39 base pairs nucleic acid single linear other nucleic acid45 CTAGTCTTCT TCTTTCTTTT TTTTTTGCGC TTCTTCTTT 39 66 base pairs nucleicacid single linear other nucleic acid 46 GATCAGGATC AGGTTCAGGGAGTGGCTCTG CCTGCGACTG TCGCGGCGAT TGTTTTTGCG 60 GTTAAG 66 66 base pairsnucleic acid single linear other nucleic acid 47 GATCCTTAAC CGCAAAAACAATCGCCGCGA CAGTCGCAGG CAGAGCCACT CCCTGAACCT 60 GATCCT 66 86 base pairsnucleic acid double linear DNA (genomic) 48 GCC CAA GAA GGA TCA GGA TCAGGT TCA GGG AGT GGC TCT GCC TGC GAC 48 Ala Gln Glu Gly Ser Gly Ser GlySer Gly Ser Gly Ser Ala Cys Asp 20 25 30 TGT CGC GGC GAT TGT TTT TGC GGTTAA GGA TCC AAT AA 86 Cys Arg Gly Asp Cys Phe Cys Gly 35 40 24 aminoacids amino acid linear peptide 49 Ala Gln Glu Gly Ser Gly Ser Gly SerGly Ser Gly Ser Ala Cys Asp 1 5 10 15 Cys Arg Gly Asp Cys Phe Cys Gly 2039 base pairs nucleic acid single linear other nucleic acid 50GATCCGGTTC AGGATCTGGC AGTGGCTCGA CTAGTTAAA 39 39 base pairs nucleic acidsingle linear other nucleic acid 51 GATCTTTAAC TAGTCGAGCC ACTGCCAGATCCTGAACCG 39 66 base pairs nucleic acid double linear DNA (genomic) 52ACT AGT GGA AGA GGA GAT ACT TTT GGC CGC GGC GAC ACG TTC GGA AGG 48 ThrSer Gly Arg Gly Asp Thr Phe Gly Arg Gly Asp Thr Phe Gly Arg 30 35 40 GGGGAT ACA TTT TCT AGT 66 Gly Asp Thr Phe Ser Ser 45 50 22 amino acidsamino acid linear peptide 53 Thr Ser Gly Arg Gly Asp Thr Phe Gly Arg GlyAsp Thr Phe Gly Arg 1 5 10 15 Gly Asp Thr Phe Ser Ser 20 60 base pairsnucleic acid single linear other nucleic acid 54 CTAGTGGAAG AGGAGATACTTTTGGCCGCG GCGACACGTT CGGAAGGGGG GATACATTTT 60 60 base pairs nucleicacid single linear other nucleic acid 55 CTAGAAAATG TATCCCCCCTTCCGAACGTG TCGCCGCGGC CAAAAGTATC TCCTCTTCCA 60 8 amino acids amino acidnot relevant linear peptide 56 Glu Ile Leu Asp Val Pro Ser Thr 1 5 21amino acids amino acid single linear peptide 57 Glu Ile Leu Asp Val ProSer Glu Ile Leu Asp Val Pro Ser Glu Il 1 5 10 15 Leu Asp Val Pro Ser 2063 base pairs nucleic acid double linear DNA (genomic) 58 ACT AGT GAAATT CTT GAC GTC GGA GAG ATC CTC GAC GTC GGG GAA ATA 48 Thr Ser Glu IleLeu Asp Val Gly Glu Ile Leu Asp Val Gly Glu Ile 25 30 35 CTG GAC GTC TCTAGT 63 Leu Asp Val Ser Ser 40 21 amino acids amino acid linear peptide59 Thr Ser Glu Ile Leu Asp Val Gly Glu Ile Leu Asp Val Gly Glu Ile 1 510 15 Leu Asp Val Ser Ser 20 57 base pairs nucleic acid single linearother nucleic acid 60 CTAGTGAAAT TCTTGACGTC GGAGAGATCC TCGACGTCGGGGAAATACTG GACGTCT 57 57 base pairs nucleic acid single linear othernucleic acid 61 CTAGAGACGT CCAGTATTTC CCCGACGTCG AGGATCTCTC CGACGTCAAGAATTTCA 57 66 base pairs nucleic acid double linear DNA (genomic) 62 ACTAGT GGA TAC ATC GGC AGT CGC GGT TAC ATT GGG TCC CGA GGA TAT 48 Thr SerGly Tyr Ile Gly Ser Arg Gly Tyr Ile Gly Ser Arg Gly Tyr 25 30 35 ATA GGCTCA AGA TCT AGT 66 Ile Gly Ser Arg Ser Ser 40 22 amino acids amino acidlinear peptide 63 Thr Ser Gly Tyr Ile Gly Ser Arg Gly Tyr Ile Gly SerArg Gly Tyr 1 5 10 15 Ile Gly Ser Arg Ser Ser 20 60 base pairs nucleicacid single linear other nucleic acid 64 CTAGTGGATA CATCGGCAGTCGCGGTTACA TTGGGTCCCG AGGATATATA GGCTCAAGAT 60 60 base pairs nucleicacid single linear other nucleic acid 65 CTAGATCTTG AGCCTATATATCCTCGGGAC CCAATGTAAC CGCGACTGCC GATGTATCCA 60 5 amino acids amino acidsingle linear peptide 66 Tyr Ile Gly Ser Arg 1 5 6 amino acids aminoacid single linear peptide 67 Tyr Ile Gly Ser Arg Gly 1 5 14 amino acidsamino acid single linear peptide 68 Ser Ala Cys Asp Cys Arg Gly Asp CysPhe Cys Cys Thr Ser 1 5 10 5 amino acids amino acid single linearpeptide 69 Ile Thr Leu Asn Gly 1 5 5 amino acids amino acid singlelinear peptide 70 Glu Ser Thr Glu Thr 1 5 7 amino acids amino acidsingle linear peptide 71 Phe Ser Tyr Ile Ala Gln Glu 1 5 10 amino acidsamino acid single linear peptide 72 Gly Ser Gly Ser Gly Ser Gly Ser GlySer 1 5 10 36 base pairs nucleic acid double linear other nucleic acid73 ATT ACA CTT AAT GGC ACT AGT GAA TCC ACA GAA ACT 36 Ile Thr Leu AsnGly Thr Ser Glu Ser Thr Glu Thr 25 30 12 amino acids amino acid linearpeptide 74 Ile Thr Leu Asn Gly Thr Ser Glu Ser Thr Glu Thr 1 5 10 105base pairs nucleic acid double linear other nucleic acid 75 GCC CAA GAAGGA TCC GGT TCA GGA TCT GGC AGT GGC TCG ACT AGT GAA 48 Ala Gln Glu GlySer Gly Ser Gly Ser Gly Ser Gly Ser Thr Ser Glu 30 35 40 ATT CTT GAC GTCGGA GAG ATC CTC GAC GTC GGG GAA ATA CTG GAC GTC 96 Ile Leu Asp Val GlyGlu Ile Leu Asp Val Gly Glu Ile Leu Asp Val 45 50 55 60 TCT AGT TAA 105Ser Ser 34 amino acids amino acid linear peptide 76 Ala Gln Glu Gly SerGly Ser Gly Ser Gly Ser Gly Ser Thr Ser Glu 1 5 10 15 Ile Leu Asp ValGly Glu Ile Leu Asp Val Gly Glu Ile Leu Asp Val 20 25 30 Ser Ser 108base pairs nucleic acid double linear other nucleic acid 77 GCC CAA GAAGGA TCC GGT TCA GGA TCT GGC AGT GGC TCG ACT AGT GGA 48 Ala Gln Glu GlySer Gly Ser Gly Ser Gly Ser Gly Ser Thr Ser Gly 40 45 50 TAC ATC GGC AGTCGC GGT TAC ATT GGG TCC CGA GGA TAT ATA GGC TCA 96 Tyr Ile Gly Ser ArgGly Tyr Ile Gly Ser Arg Gly Tyr Ile Gly Ser 55 60 65 AGA TCT AGT TAA 108Arg Ser Ser 70 35 amino acids amino acid linear peptide 78 Ala Gln GluGly Ser Gly Ser Gly Ser Gly Ser Gly Ser Thr Ser Gly 1 5 10 15 Tyr IleGly Ser Arg Gly Tyr Ile Gly Ser Arg Gly Tyr Ile Gly Ser 20 25 30 Arg SerSer 35 12 amino acids amino acid single linear peptide 79 Ser Ala CysAsp Cys Arg Gly Asp Cys Phe Cys Gly 1 5 10 7 amino acids amino acidsingle linear peptide 80 Glu Ile Leu Asp Val Pro Ser 1 5

What is claimed is:
 1. A chimeric adenovirus fiber protein comprising anonnative amino acid sequence that is constrained by a preexisting loop.2. The chimeric adenovirus fiber protein of claim 1, which directs entryinto cells of a vector comprising said chimeric adenovirus fiber proteinthat is more efficient than entry into cells of a vector that isidentical except for comprising a wild-type adenovirus fiber proteinrather than said chimeric adenovirus protein.
 3. The chimeric adenovirusfiber protein of claim 1, which binds a binding site present on a cellsurface which wild-type fiber protein does not bind.
 4. The chimericadenovirus fiber protein of claim 1, wherein said normative amino acidsequence comprises an epitope for an antibody or a ligand for a cellsurface binding site.
 5. The chimeric adenovirus fiber protein of claim1, wherein said nonnative amino acid sequence comprises from about 3 toabout 200 amino acids.
 6. The chimeric adenovirus fiber protein of claim1, wherein said nonnative amino acid sequence comprises from about 3 toabout 30 amino acids.
 7. The chimeric adenovirus fiber protein of claim1, wherein said nonnative amino acid sequence comprises an RGD sequence.8. The chimeric adenovirus fiber protein of claim 1, wherein saidnonnative amino acid sequence comprises a sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:31, SEQID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:53, SEQ IDNO:56, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, and SEQ ID NO:79, and wherein said sequence may be deleted ateither the C- or N-terminus by 1, 2, or 3 residues.
 9. The chimericadenovirus fiber protein of claim 8, wherein said sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5.
 10. Thechimeric adenovirus fiber protein of claim 1, wherein said nonnativeamino acid sequence comprises a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:31, SEQ IDNO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:53, SEQ IDNO:56, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, and SEQ ID NO:79, and conservative amino acid substitutionsthereof.
 11. The chimeric adenovirus fiber protein of claim 10, whereinsaid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5.
 12. An adenovirus comprising the chimeric adenovirusfiber protein of claim
 1. 13. The adenovirus of claim 12, furthercomprising a genome comprising a passenger gene.
 14. A host cellcomprising the adenovirus of claim
 12. 15. A nucleic acid encoding theprotein of claim
 1. 16. A vector comprising the nucleic acid of claim 1.17. The vector of claim 16, wherein said vector derived from a virus.18. The vector of claim 16, wherein said vector is an adenoviral vector.19. The vector of claim 16, wherein said vector further comprises apassenger gene.
 20. A host cell comprising the vector of claim 16.